589.95 
W15b 

,.,,.iaa:-ff 


A.B:s.-rRA;fi: 


BACTERIOLOGY 

IN 

ABSTRACT 

BY 

A.  B.  WALLGREN.  B.  S..  M.  D. 

Assistant  Professor  of  Biology,  University  of 
Pittsburgh;  Pathologist  to  the  Pittsburgh 
Hospital,  St.  Margaret's  Hospital  and 
Columbia  Hospital;    Author  of 
Histology  in  Abstract  and 
Pathology  in  Absti  ict 

PITTSBURGH,  PENNA. 


Published  by 
Medical  Abstract  Publishing  Company 
Jenkins  Arcade  Building 
Pittsburgh,  Pa. 


PREFACE  TO  SECOND  EDITION 


The  demand  created  by  the  first 
Abstract  in  Bacteriology  necessitated  the 
production  of  a  second  edition,  which,  while 
considerably  enlarged,  contains  only  the 
very  elements  of  Bacteriology.  Although 
containing  only  the  first  principles  of 
Bacteriology,  it  is  hoped  that  this  book 
will  be  of  use  to  the  student  wishing  a 
small  work  for  a  ready  guide. 

For  a  detailed  work  the  student  must 
necessarily  refer  to  the  works  of  Jordan, 
Mallory  and  Wright,  Chester,  Frost,  Frost 
and  McCampbell,  Marshall,  Park  and 
Williams,  Hiss  and  Zinsser,  Besson, 
Gorham,  Muir  and  Ritchie,  Simon,  etc., 
all  of  which  have  been  freely  consulted  in 
the  preparation  of  this  Abstract. 

The  Author  is  very  much  indebted  to 
Eleanor  C.  Doty  for  valuable  aid  m  the 
preparation  of  manuscript  and  the  reading 
of  proofs. 


5  ^9,  9  6^ 

\A/  /6'  b 
history"  OF  BACTERIOLOGY. 

^      With  the  introduction  of  the  micro- 
J  scope    some    order    was    brought  into 
S  understanding-  that,  group  of  organisms 
^  which    Linnaeus    had    termed  "chaos." 
^  That  disease  and  decay   were   due  to 
\  minute  organisms  had  been  the  theory 
S  for  centuries.     The  conception  of  con- 
\,  tagion,  or  the  transmission  of  disease 
^  from  one  human  being  to  another,  was 
'  centuries  old.    This  fact  had  been  rec- 
ognized by  Aristotle  and  had  been  re- 
iterated by  medieval  philosophers,  and 
^  had  led  to  the  division  of  contagious 
^  diseases,  by  Pracastor  (1546),  into  those 
diseases    transmitted    "per  contactum" 
-  and     those    conveyed    indirectly  "per 
i  fomitem."    It  was  on  account  of  these 
k  facts  of  transmissibility  of  disease  that 
^  physicians    of    the    eighteenth  century 
saw  an  explanation  in  the  microorgan- 
\  isms  discovered  in  the  latter  half  of  the 
;  seventeenth     century    by     the  Jesuit, 
Kircher    (1659),   and   the   Dutch  linen- 
draper,  van  Leeuwenhoek  (1675).  These 
two   men   were   able   by   the  improved 
microscope  to  demonstrate  microorgan- 
isms in  water,  intestinal  contents,  etc. 
They    made    out    short,    straight,  and 
curved  rods,  and  described  their  motil- 
ity.    There  can  be  no  doubt  that  the 
bodies  seen  by  these  two  men  were,  at 
least  in  part,  bacteria.    During  the  cen- 
tury following  the  work  of  these  two 
investigators,  a  more  exact  description 
^of   these  forms   of  life  led   Muller  to 
attempt  a  systematic  classification.  A 
^more  extensive  study  and  classification 
^was  later  made  by  Ehrenburg. 

Needham,     in     1749,     published  an 
^^rticle  in  which  he  favored  the  opinion, 
^held     by     many,     namely,     that  the 
^minute  organisms  described  by  Leeuw- 
enhoek  and   others   were   produced  by 
t^tepontaneous    generation.      He    held  to 
.this  opinion  from  the  fact  that  he  had 
placed    putrefying   material    and  vege- 
\table  infusion  in  sealed  flasks,  exposing 
Pthem  for  a  short  time  to  heat,  and  later 
y^ound   this   infusion   to   be   filled  with 
-^^microorganisms.    BufCon  supported  him 
"^in  his  views.     Abbe'  Spallanazanni  re- 
peated   the    experiments    of  Needham, 
employing  greater  care  in  sealing  his 
flasks,  and  subjecting  them  to  a  greater 
exposure  of  heat.    His  results  were  un- 


4  BACTERIOLOGY. 

like  those  of  Needham  and  consequently 
did  not  support  the  theory  of  spon- 
taneous generation.  Schulze's  (1836) 
failure  to  find  a  living  organism  in  in- 
fusions which  had  been  boiled  and  to 
which  air  had  been  admitted  only  after 
passage  through  acid  solutions,  did  not 
convince  many  that  spontaneous  gener- 
ation was  impossible,  and  that  the  life 
that  appeared  in  the  infusions  was  due 
to  the  presence  of  minute  organisms 
having  entered  the  infusion  by  reason 
of  faulty  technique.  The  question  of 
spontaneous  generation  was  not  definite- 
ly settled  until  Pasteur  (1860)  conduct- 
ed a  series  of  experiments,  the  results 
of  which  absolutely  refuted  the  doctrine 
of  spontaneous  generation. 

Plenciz  of  Vienna  (1762),  expressed 
his  belief  in  the  direct  etiological  con- 
nection between  microorganisms  and 
certain  diseases,  and  also  suggested 
specific  treatment  for  these  diseases. 

Rayer,  in  1815,  found  rod  shaped  or- 
ganisms in  the  blood  of  animals  sick 
with  splenic  fever.  The  real  advance 
in  the  development  of  bacteriology 
came  in  1837  when  Schwann  showed 
that  yeasts  were  living  organisms  and 
had  to  do  with  the  process  of  fermen- 
tation. The  same  view  was  held  by  the 
Frenchman  Cagniard-Latour. 

Pasteur  paralleled  his  researches  up- 
oti  spontaneous  generation  with  experi- 
ments upon  fermentation  along  the 
lines  suggested  by  Cagniard-Latour, 
publishing  his  classical  studies  upon 
the  fermentation  that  occurred  in  beer 
and  wine,  due  to  yeasts,  and  was  also 
able  to  show  that  a  number  of  other 
fermentations,  such  as  those  of  lactic 
and  butyric  acid,  as  well  as  the  decom- 
position of  organic  matter  by  putrefac- 
tion, were  directly  due  to  the  action  of 
microorganisms.  The  doctrine  of  spon- 
taneous generation  had  received  its  final 
refutation  in  all  but  one  particular.  It 
was  not  understood  why  sterility  could 
not  always  be  obtained  from  the  appli- 
cation of  definite  degrees  of  heat.  This 
was  finally  explained  by  Cohn  (1871), 
who  demonstrated  the  presence  of  bac- 
terial spores,  also  demonstrating  their 
resistance  toward  heat  and  other  influ- 
ences. 

Pollender,  in  1855,  reported  the  pres- 
ence of  rod  shaped  bodies  in  the  blood 


BACTERIOLOGY.  5 

and  spleen  of  animals  dead  of  anthrax, 
previously  reported  by  Rayer.  Convinc- 
ing proof  of  the  observations  of  Rayer 
and  Pollender  was  .  brought  out  by 
Davaine  (1863),  who  also  succeeded  in 
demonstrating  that  anthrax  could  be 
transmitted  by  means  of  blood  contain- 
ing the  rods  described  by  Rayer  and 
Davaine,  and  could  never  be  transmitted 
by  blood  from  which  the  rods  were  ab- 
sent. Anthrax,  therefore,  was  the  first 
disease  in  which  bacterial  causation 
was  demonstrated. 

Obermeier  (1868)  demonstrated  the 
presence  of  a  spirillum  in  the  blood  ot 
patients  suffering  from  relapsing  fever. 

Rindfleisch,  von  Recklinghausen,  Wal- 
deyer  described  microorganisms  in  tis- 
sues containing  abscesses. 

Klebs  (1870)  described  similar  mi- 
croorganisms found  in  pus. 

Koch  (1880)  introduced  nutrient  me- 
dia upon  which  bacteria  could  be  cul- 
tured, thus  laying  the  foundation  for 
an  exact  science. 

Weigert,  Koch,  and  Ehrlich  intro- 
duced the  use  of  aniline  dyes  which 
facilitated  the  morphological  study  of 
bacteria. 

Koch  published  the  discovery  of  the 
typhoid  bacillus,  fowl  cholera  bacillus 
and  pneumococcus  in  1880  and  the 
tubercle  bacillus  in  1882. 

THE  BIOLOGY  OF  BACTERIA. 

ORIGIN,  DISTRIBUTION,  ETC. 

Bacteria  cannot  arise  denovo.  They 
must  develop  from  pre-existing  bacteria 
or  their  spores.  One  kind  of  organism 
will  not  produce  another  kind. 

DISTRIBUTION. 

Air.  In  the  air  there  are  bacteria 
throughout,  except  at  great  altitudes. 

Soil.  In  soil,  bacteria  are  very  abund- 
ant except  at  great  depths.  Garden 
soil  will  contain  from  a  hundred 
thousand  to  as  many  millions  per  gm. 
Bacteria  are  less  numerous  in  the 
peat  or  acid  moor  soils,  in  the  soils 
containing  an  excess  of  alkali  and  in 
sandy  soils.  Were  it  not  for  bac- 
teria, plant  life  could  not  exist 
as  they  perform  functions  in  the  soil, 


6  BACTERIOLOGY. 

fitting  it  physically  and  chemically 
for  the  growth  of  higher  plants. 
They  decompose  organic  matter  di- 
rectly, and  minerals  of  the  soil  in- 
directly, converting  them  into  com- 
pounds that  are  taken  up  and  assim- 
ilated by  plants,  etc. 
"Water,  as  a  rule,  is  the  home  of  bac- 
teria. 

Wells  contain  from  a  very  few  to 
several  hundred  bacteria  per  c.  c, 
though  in  deep  wells,  such  as  arte- 
sian, and  in  the  springs  of  some  dis- 
tricts, there  may  be  no  bacteria. 
There  are  about  the  same  number  of 
bacteria  in  lakes  and  ponds  as  in 
wells,  except  where  there  is  sewage 
contamination. 

Streams  usually  contain  more  bac- 
teria than  any  of  the  above  by  rea- 
son of  sewage  contamination  or  wash- 
ings from  water  sheds,  etc. 
Foods.  Bacteria  are  most  abundant  in 
most  foods.  Some  healthful  foods 
contain  great  numbers  of  bacteria,  as 
is  illustrated  by  the  lactic  acid  bac- 
teria of  sour  milk. 

Other  bacteria  present  in  food,  in 
great  numbers,  may  be  harmless. 

The  bacteria  of  foods  may  be  di- 
vided into  three  groups: — 

(1)  Those     beneficial — which     bring  # 

about  the  fermentations,  as  in 
the  preparation  of  pickles, 
sauerkraut,  etc. 

(2)  Those    producing  fermentations 

and  decay. 

(3)  Those  producing  disease. 
Fermentation     bacteria     act  upon 

carbohydrates,   as    starch   and  cellu- 
lose,   breaking    them    down.  Decay- 
bacteria  decompose  proteins. 
Body.    The  normal  living  tissues  of  the 
body  are  free  from  bacteria. 

Bacteria  are  found  in  great  num- 
bers on  the  skin,  in  the  alimentary 
tract,  and  can  be  considered  as  a 
flora  peculiar  to  this  situation  as  they 
ordinarily  do  no  harm.  Certain  bac- 
teria (pathogenic)  may  enter  the  body 
and  produce  disease. 

MICROORGANISM  NORMAL  TO  THE 
HUMAN  BODY. 

A  great  number  of  species  of  bacteria 
develop  on  the  skin  of  the  body  and 


BACTERIOLOGY. 


7 


in  the  various  body  cavities  which 
open  to  the  surface.  Normally  the  tis- 
sues of  the  body  are  sterile.  Bacteria 
are  not  found  in  the  blood  stream,  in 
the  muscles,  nor  in  the  glands  oi  nor- 
mal individuals  although  the  lymph 
fluids  lying-  near  the  intestines  may 
occasionally  be  infected  by  the  intest- 
inal bacteria.  Organisms  may  or  may 
not  be  present  in  the  tissues  of  the 
individuals.  Some  diseases  are  classi- 
fied as  septicemias  and  bacteriemias  be- 
cause of  the  general  distribution  of  the 
bacteria  in  the  blood  stream.  In  other 
cases,  the  organisms  are  localized  and 
do  not  get  far  from  the  site  of  infec- 
tion. 

The  exposed  surfaces  of  the  body  and 
the  cavities  of  the  body  that  communi- 
cate with  the  surface  possess  a  bac- 
terial flora  that  may  be  considered 
normal  to  the  part. 
The  Flora  of  the  Skin. 

The  organisms  that  are  constantly 
present  and  evidently  multiply  upon 
the  surface: — 

Micrococcus  (staphylococcus)  epider- 
midosis  albus,  an  organism  which 
is  closely  related  to  the  pus  pro- 
ducing cocci.  This  organism  fre- 
quently penetrates  into  the  lower 
layers  of  the  epidermis. 

Micrococcus  aureus  and  albus  are  fre- 
quently present,  but  are  usually  of 
a  low  virulence.  These  are  the  or- 
ganisms that  produce  infection  of 
wounds,  abscesses,  and  boils. 

Streptococci  may  frequently  be  iso- 
lated from  the  skin. 

Bacterium  smegmatis  is  nearly  con- 
stantly present  in  places  where  the 
skin  is  moist  and  greasy.  It  does 
not  seem  to  penetrate  into  the  skin 
but  lives  upon  the  skin  excreta. 

In  the  wax  secreted  in  the  external 
auditory  canal,  the  micrococcus 
cereus  flavus  is  generally  found. 

Very  few  bacteria  are  found  normal- 
ly on  the  conjunctiva.  The  tears 
seem  to  remove  or  destroy  any  or- 
ganism that  may  gain  entrance. 

The  organisms  that  accidentally  form 
a  skin  flora  are  the  bacteria  that 
contaminate  the  skin  when  it  comes 
into  contact  with  dust  or  dirt.  The 
soil  bacteria  are  quite  common  to 
the    skin,    particularly    under  the 


8  BACTERIOLOGY. 

finger  nails  and  in  the  hair.  These 
include  the  soil  aerobic  forms,  as 
the  bacillus  subtilis  group  and  cer- 
tain anaerobic  bacteria. 

The  intestinal  organisms,  as  the  ba- 
cillus  coli,   are   commonly  present 
by  reason  of  contact  with  diseased 
individuals. 
The  Flora  of  the  Mouth. 

A  great  number  of  tne  organisms  that 
have  been  isolated  from  the  mouth 
are  probably  due  to  accidental  con- 
tamination. Mueller  has  described 
a  number  of  species  found  present 
in  the  normal  mouth.  Of  these,  he 
decided  that  six  were  constantly 
present;  i.  e.,  leptothrix  and  the 
spirocheta  dentium.  These  organ- 
isms are  obligative  anaerobes  and 
find  favorable  conditions  for  growth 
between  the  teeth  and  in  teeth  un- 
dergoing decay. 

Anaerobic  bacilli  related  to  B.  putri- 
ficus. 

Non  virulent  varieties  of  streptococ- 
cus pyogenes. 

Non  virulent  varieties  of  micrococcus 
aureus  and  albus. 

Avirulent  pneumococcus. 

The  decay  of  teeth  is  said  to  be  due 
to  the  development  of  acids  by  cer- 
tain lactic  acid  bacteria  present 
upon  the  surface,  causing  a  more  or 
less  complete  removal  of  the  lime 

*  present;  while  the  decomposition  of 
the  remaining  substance  of  the 
teeth  is  probably  brought  about  by 
common  anaerobic  bacteria.  Occa- 
sionally the  mouth  of  a  healthy  in- 
dividual may  contain  pathogenic 
organisms,  as  the  bacterium  diph- 
theria and  bacterium  infiuenza. 
The  Flora  of  the  Stomach. 

Ordinarily  bacteria  do  not  develop  in 
the  stomach  by  reason  of  its  acid- 
ity. If  for  any  reason  the  acidity 
is  diminished,  bacteria  belonging  to 
the  butyric  acid  group  and  the  bac- 
terium lactis  aerogenes  may  de- 
velop. Under  certain  conditions, 
the  lactic  acid  bacteria  of  the  Bul- 
garian type  may  develop. 
Flora  of  Intestine. 

The  first  portion  of  the  intestine  is 
relatively  free  from  bacteria  by 
reason  of  the  fact  that  the  bile  is 
antiseptic  to  certain  microorganisms 


BACTERIOLOGY. 


9 


and  will,  more  or  less,  completely 
inhibit  their  growth.  In  the  lower 
part  of  the  small  intestine  bacteria 
become  more  numerous.  The  bac- 
terium lactis  aerogenes  and  bacil- 
lus coli  are  present  in  small  num- 
bers. In  the  colon,  bacteria  develop 
freely.  The  bacterium  lactis  aero- 
geneus  and  the  bacillus  coli  are 
nearly  always  present.  These  to- 
gether with  the  bacillus  bifRdus  and 
bacillus  acidophilus  inhibit  the 
growth  of  any  organism  of  the 
putrefactive  type  by  the  formation 
of  an  acid,  and  possibly  by  the  for- 
mation of  metabolic  products.  An- 
aerobic forms,  as  the  bacillus  putrif- 
icus  and  bacillus  aerogenes  capsul- 
atus,  are  also  present. 

It  is  quite  possible  that  in  some  of 
the  herbivorous  animals  the  organ- 
isms are  of  some  assistance  in  the 
digestion  of  food.  In  the  human 
intestine  it  is  believed  that  products 
of  decomposition  brought  about  by 
the  putrefactive  bacteria  are  ab- 
sorbed through  the  intestinal  wall 
and  appear  in  the  same  form  or  as 
related  compounds  in  the  urine,  and 
cause  some  of  the  changes  charac- 
teristic of  old  age,  particularly  the 
hardening  of  the  arterial  wall 
(arterial  sclerosis). 

Metchnikofe  believed  that  the  putre- 
factive bacteria  might  be  elimin- 
ated by  using  foods  containing 
great  numbers  of  lactic  acid  bac- 
teria, which  would  prevent  the  de- 
velopment of  the  putrefactive  form 
by  the  presence  of  the  lactic  acid 
bacteria  and  also  the  lactic  acid. 
The  Plora  of  the  Respiratory  Tract. 

The  flora  of  the  upper  respiratory 
passages,  as  the  nose,  can  not  be 
said  to  be  characteristic.  The  bac- 
teria isolated  from  the  nasal  cav- 
ities are  generally  the  bacteria  of 
the  air.  The  air  of  the  bronchi,  the 
bronchioles  and  alveoli  of  the  lungs 
are  usually  entirely  free  from  mi- 
croorganisms by  reason  of  the  filtra- 
tion of  air  through  the  nasal  pass- 
ages, and  the  passage  of  the  air 
over  the  nasal  and  oral  mucous 
membrane,  which  is  covered  with 
mucous  and  also  with  ciliated  cells, 
which  serve  to  some  extent  to  wash 


10  BACTERIOLOGY. 


the  mucous  containing  the  bacteria 
from  the  surface.  Infections  of 
the  nasal  mucous  membrane  are, 
however,  not  uncommon.  The  bac- 
terium influenza,  the  streptococcus 
pyogenes,  the  micrococcus  pyogenes, 
aureus  and  albus,  bacterium  diph- 
theria, the  micrococcus  intracel- 
lularis,  meningitidis  and  occasion- 
ally the  bacterium  mallein  produce 
infection  through  the  membrane  of 
the  nasal  cavities. 
The  Flora  of  the  Genito-Urinary  Tract. 

The  exposed  mucous  surfaces  of  the 
genito-urinary  tract  usually  harbor 
a  number  of  harmless  bacteria. 
Organisms  closely  related  to  the  pus 
producing  cocci  are  commonly  pres- 
ent. The  secretions  of  the  vagina 
are  somewhat  bactericidal,  but  cer- 
tain bacteria  are  constantly  present 
and  the  properties  of  the  vaginal 
secretion  is  supposed  to  be  in  part 
due  to  the  presence  of  these  organ- 
isms, e.  g.     Doederlein's  bacillus. 

Normally  the  uterus  does  not  contain 
bacteria,  although  their  presence  is 
not  uncommon  in  the  later  stages 
of  pregnancy.  The  urinary  bladder 
is  normally  free  from  bacteria. 

In  the  secretions  about  the  external 
genitals,  acid-fast  bacteria,  partic- 
ularly the  bacterium  smegmatis,  are 
constantly  present. 

THE  CATABOLIC  ACTIVITIES  OF  BACTERIA 

Study  of  sulistances  that  result  from 
the  action  of  the  life  of  bacteria  and 
the  changreb  that  they  produce  in  the 
various  media  of  growth,  is  really  a 
branch  of  organic  chemistry.     So  we 
can  only  make  mention  of  them  here. 
The  function  of  bacteria  is  essentially 
a  destructive  one.    They  split  up  the 
higher   nitrogenous   and  non-nitro- 
genous compounds  into  simple  sub- 
stances. 

The  various  substances  that  are  found 
in  the  media  of  bacterial  growth 
comprise : — 

1.  The  components  of  the  bacterial 

cell  proper,  as  the  proteins. 

2.  The  secretions  of  the  cell,  as  the 

ferments  and  toxins. 

3.  The  substances  that  are  the  re- 

sult of  the  action  of  the  organ- 


BACTERIOLOGY. 


11 


isms  upon  the  medium  of 
growth: — 
The  toxic  substances  in  bacterial 
cultures  may  be  classified  as 
(a).  Intracellular  and  (b)  Ex- 
tracellular, according-  as  they 
are  contained  within  the  bac- 
terial cell  or  free  in  the  culture 
medium.  The  extracellular  sub- 
stances may  be  purely  products 
of  bacterial  secretion  which 
have  separated  from  the  cell  or 
they  may  be  decomposition 
products  derived  from  the  cul- 
ture meuium. 

(1)  The  proteins  may  produce  sup- 
purations or  fever,  or  may 
cause  inflammatory  processes. 
They  are  comparatively  resist- 
ant to  heat  and  are  thus  sharply 
distinguished  from  the  ferments 
and  toxins.  The  best  known 
examples  are  mallein,  derived 
from  the  bacillus  of  glanders, 
and  tuberculin  from  that  of 
tuberculosis.  These  substances 
are  pyogrenlc  (fever)  when  in- 
jected into  animals  sick  with 
glanders  or  tuberculosis  but 
have  very  slight  effect  upon 
healthy  subjects. 

(2)  Second  group  are  ferments  and 
possibly  toxins. 

Ferments  are  complex  bodies 
about  which  very  little  is 
known,  except  the  effects  they 
produce.  By  their  presence  and 
probably  without  entering  into 
intimate  chemical  combination, 
they  possess  the  power  of 
breaking  up  more  highly  organ- 
ized nitrogenous  and  non-nitro- 
genous compounds  into  simple 
and  more  diffusible  molecules. 
They  are  termed  enzymes  or  un- 
formed ferments  in  contradis- 
tinction to  the  bacteria  them- 
selves. That  the  action  of  fer- 
ments is  not  due  directly  to  the 
organism  is  shown  by  the  fact 
that  bactericidal  substances, 
such  as  phenol  5%,  chloroform, 
ether,  etc.,  have  no  effect  on 
them  and  that  cultures  freed 
from  bacteria  by  filtration  still 
possess  fermentative  power. 


BACTERIOLOGY. 

The  action  of  ferments  is  termed 
fermentation,  but  this  term  is 
more  especially  limited  to  the 
effect  of  certain  ferments  upon 
non-nitrogenous  compounds,  par- 
ticularly the  carbohydrates.  The 
result  of  fermentation  upon  ni- 
trogenous material  is  called 
putrefaction,  which  generally 
occurs  with,  though  often  with- 
out, the  formation  of  odorous 
gases  and  other  substances. 

The  intracellular  origin  of  certain 
ferments  has  been  demon- 
strated by  their  experimental 
separation  from  the  bacteria 
when  placed  under  high  pres- 
sures. The  resulting  bacteria- 
free  liquid  possesses  the  same 
fermenting  qualities  as  the  cul- 
ture itself. 

Ferments  like  toxins  are  of  un- 
known composition,  are  highly 
destructible  by  chemical  agents 
and  heat,  and  cause  effects  out 
of  all  proportion  to  their  bulk 
or  amount.  They  are  frequent- 
ly mechanically  precipitated 
with  various  indifferent  bodies. 
When  injected  into  animals 
both  are  capable  of  exciting  the 
formation  of  antibodies  (anti- 
ferments  and  antitoxins). 
The  principal  ferments  are: — 

Proteolytic  ferments.  Transform- 
ing albumins  into  more  soluble 
and  diffusible  substances. 

These  ferments  find  their  analogy 
in  the  ferments  of  the  stomach 
and  pancreas. 

They  digest  the  various  albumins 
with  the  formation  of  album- 
oses  and  those  end  products  of 
hydrolysis  which  are  collective- 
ly spoken  of  as  peptones. 

One  form  very  often  met  with 
liquefies  gelatin.  It  acts  in 
an  alkaline  medium  and  is 
therefore  akin  to  the  animal 
ferment  trypsin.  This  lique- 
faction of  gelatin  affords  a 
means  of  distinguishing  many 
species  of  organisms. 

Amylolytic  ferments  (amy  las  is 
diastases)  transform  starches 
into  sugar  and  are  found  in 
many  bacterial   cultures,  e.  g., 


BACTERIOLOGY.  13 

B.  mallei,  B.  pneumonia,  etc. 
They  find  their  analogy  in  the 
secretions  of  the  pancreas  and 
the  ptyalin  of  saliva. 
InvertlniT  ferments  (Invertases.) 
They  are  apparently  related  to 
the  amylolytic  ferments  and 
are  to  a  certain  extent  iden- 
tical with  them.  They  invert 
disaccharides  to  monosacchar- 
ides and  according-  to  their  spe- 
cific action  upon  cane  sugar, 
maltose  and  lactose,  they  are 
termed  —  invertases,  maltases 
and  lactases,  respectively.  They 
have  their  analogies  in  the  sa- 
liva and  pancreatic  juice.  Such 
ferments  are  found  in  cultures 
of  spirillum  cholera  and  Metch- 
nikovi. 

Emulsifying'  Ferments  are  formed 
by  but  few  bacteria.  One  ex- 
ample is  micrococcus  pyogenes 
tenuis. 

Coag'ulating'  Ferments.  In  these 
we  have  a  means  of  differenti- 
ating bacteria  by  their  co- 
agulation of  milk.  This  coagu- 
lation is  not  due  to  acids  pro- 
duced in  the  medium  but  to  the 
action  of  a  ferment.  Some 
varieties  of  bacteria  produce  a 
ferment  that  has  the  power  of 
dissolving  this  coagulum  when 
formed  (casease).  Other  bac- 
teria produce  both  ferments — 
the  coagulating  and  the  dis- 
solving. 

Jjipolytic  Ferments.  Fat  splitting 
ferments  (lipases). 

They  cause  hydrolysis  of  fats  into 
fatty  acids  and  glycerin.  They 
have  their  analogues  in  the 
pancreatic  and  gastric  juice. 

Ureases  (hydrolytic)  break  up 
urea  into  ammonium  carbonate 
and  hippuric  acid  into  glycocol 
and  benzoic  acid.  They  are  in 
such  bacteria  as  the  micrococ- 
cus urea,  bacterium  urea,  bacil- 
lus fluorescens,  etc. 

Oxidases.  Produced  by  several 
bacteria,  especially  the  chrom- 
ogens.  They  act  upon  complex 
organic  compounds  changing 
them  to  colored  bodies. 


14  BACTERIOLOGY. 


Beductases.  This  ferment  is  con- 
tained by  all  but  a  few  bacteria, 
among-  the  exceptions  being  the 
Bact.  acidi  lactici.  They  will 
reduce  nitrates  to  nitrites,  sul- 
phur to  hydrog-en  sulphide,  etc. 

Effects  of  Perments.  The  single 
or  combined  action  of  these 
various  ferments  cause  cer- 
tain kinds  of  fermentation 
distinguished  by  the  principal 
substance  produced.  Alcoholic, 
lactic  acid,  and  butyric-acid 
fermentation  of  the  sugars, 
acetic  acid  fermentation  of  al- 
cohol (B.  acidi  lactice.  B.  buty- 
ricus,  B.  acidi  butyrici,  B. 
aceticus,  etc.);  cellulose  fer- 
mentation with  the  production 
of  carbonic  acid  gas  and  am- 
monia; nitrification,  in  which 
oxidation  of  ammonium  leads  to 
the  production  of  nitrites  and 
secondarily,  conversion  of  ni- 
trites into  nitrates;  mucoid  fer- 
mentation of  grlucose  and  invert 
sugar  are  examples. 

Toxins  will  be  taJien  up  later. 

The  study  of  those  substances  result- 
ing from  the  actions  of  the  life  of 
bacteria  in  the  media  of  growth,  is 
accomplished   by    the  so-called 

Biochexuical  Methods,  1.  e.. 

Test  the  cultivations  of  the  or- 
ganism for  the  presence  of — 

1.  Soluble  enzymes — protiolytic,  di- 

astic,  invertase. 

2.  Org'anic  acids — (a).  Quantitative- 

ly— i.  e.  Estimate  the  total 
acid  production.  (b).  Qual- 
itatively, for  formic,  acetic, 
propionic,  butyric  and  lactic. 

3.  Ammonia. 

4.  Neutral     Volatile     substances — 

ethyl,  alcohol,  aldehyde,  acetone. 

5.  Aromatic      products  —  Indol  — 

phenol. 

6.  Soluble  pigrments. 

7.  Beducingr    Powers     (a)  coloring 

matters,   (b)  nitrates  to  nitrites. 

8.  Gas  production — H2    S.,    CO2  H. 

Estimate  the  ratio  between  the 
last  two  ^ases. 
Prepare    all    cultivations    for  these 
methods     of     examination  under 
optimum  conditions,  previously  de- 


BACTERIOLOGY.  15 

termined  for  each  of  the  organisms 
it  is  intended  to  investigate  as  to — 

(a)  Reaction  of  medium. 

(b)  Incubation  temperature. 

(c)  Atmospheric  environment. 
Keep  careful  records  of  these  points, 

also  the  age  of  the  cultivation  used 
in  the  final  examination. 

Examine  the  cultivations  for  the  var- 
ious products  of  bacterial  metabol- 
ism after  48  hours  growth,  and 
never  omit  to  examine  "control" 
(uninoculated)  tulbe  or  flask  of 
medium  from  the  same  Ibatch,  kept 
for  a  similar  period  under  identical 
conditions. 

If  the  results  are  negative,  test  fur- 
ther cultivations  at  3,  5  and  10  days. 

CHEMICAL  CONSTITUENTS  OF 
THE  BACTERIAL  CELL. 

The  chemical  composition  of  bacteria 
varies  with  their  food  supply. 

About  85%  of  the  bacterial  body  is  water. 
The  remainder  is  chiefly  proteids 
which  constitutes  from  50  to  85%  of 
the  dry  substance.  After  the  proteid 
material  has  been  extracted  there  are 
fats  and  in  some  instances  true  wax 
(fatty  acid  combinations  with  higher 
alcohols.  In  traces  of  cellulose  and 
ash  the  ash  constitutes  about  1  to  2% 
of  the  dry  substance,  and  is  made  up 
largely  of  phosphates  and  chlorides 
of  potassium,  sodium,  calcium  and 
magnesium. 

NUTRITION  OF  BACTERIA. 

In  order  that  bacteria  may  develop  and 
multiply,  they  must  be  supplied  with 
the  substances  of  which  they  consist, 
in  proper  quantity  and  in  forms  suit- 
able for  assimilation,  namely,  carbon, 
oxygen,  hydrogen,  nitrogen,  inorganic 
salts  and  varying  quantities  of  phos- 
phorus and  sulphur. 

Carbon  is  necessary  for  their  nourish- 
ment, and  may  be  obtained  from 
proteids,  carbohydrates  and  fats,  or 
from   their  derivatives. 

Ozygren.  Free  oxygen  is  necessary  for 
the  growth  of  many  bacteria 
(obligatory  aerobes).  For  these  it  is 
obtained  directly  from  the  atmosphere 
in  the  form  of  free  O.  Another  class 
of  bacteria  is  unable  to  develop  in  the 


16  BACTERIOLOGY. 

presence  of  free  oxygen  (obligatory- 
anaerobes),  and  they  obtain  their 
oxygen  indirectly  by  the  enzymatic 
processes  of  fermentative  and  proteo- 
lytic cleavage,  from  carbohydrates 
and  proteids,  or  by  reduction  from 
reducible  bodies. 

There  is  still  another  large  group  of  bac- 
teria which  develops  well  under  both 
aerobic  and  anaerobic  conditions. 
Some  of  these  have  a  preference  for 
free  oxygen,  but  will  thrive  without 
it  (facultative  anaerobes).  In  others 
the  reverse  is  true  (facultative 
aerobes.) 

Nitrosren  is  in  most  cases  obtained  from 
proteids.  The  diffusible  proteids  are 
the  most  important,  but  many  of  the 
non-diffusible  albumins  may  be  rend- 
ered assimilable  by  the  proteolyzing 
enzymes  possessed  by  many  bacteria. 

A  large  number  of  bacteria  may  develop 
on  media  containing  no  proteid,  in 
which  case  the  organism  produces  a 
synthetic  proteid. 

Many  bacteria  may  obtain  their  nitrogen 
from  creatin,  creatinin,  urea  and 
urates  and  ammonia  compounds  and  ni- 
trates. 

A  few  bacteria,  as  the  bacilli  in  the  root 
tubercles  of  legumins  and  the  nitro- 
gen-fixing bacteria  of  the  soil,  obtain 
their  supply  of  nitrogen  directly  from 
the  free  N  of  the  air. 

Hydrogren  is  obtained  in  combination  as 
water  and  together  with  the  carbon 
and  nitrogen  containing  substances. 

Salts.  The  phosphates  are  necessary 
constituents  of  culture  media  and  are 
taken  in  as  phosphates  of  magnesium, 
calcium,  sodium  or  potassium. 

The  chlorides  are  not  absolutely  essen- 
tial (Proskener  and  Beck). 

The  sodium  salts  seem  better  for  pur- 
poses of  cultivation  than  potassium 
salts. 

The  sulphur  is  usually  taken  in  as 
soluble  sulphates.  The  thiobacteria 
of  Winogradsky  demand  free  H2  S. 

The  iron  in  the  higher  bacteria  is  taken 
in  as  ferrous  compounds  and  is  oxid- 
ized in  the  bacterial  body  into  ferric 
compounds. 


BACTERIOLOGY.  17 

BIOLOGIC  ACTIVITIES  OF  BACTERIA. 

Without  the  bacterial  processes,  which 
are  constantly  active  in  the  reduction 
of  complex  organic  substances  to  their 
simpler  compounds,  the  chemical  in- 
terchange between  the  animal  and 
vegetable  kingdom  would  fail  and  all 
life  would  cease. 

They  are  paramount  factors  in  the  cycle 
of  living  matter  supplying  the  links 
in  the  constant  circulation  of  nitrogen 
and  carbon  compounds  necessary  be- 
tween the  plant  and  the  animal  king- 
doms, through  their  anabolic  or  con- 
structive process  in  the  one  and  their 
catabolic  or  destructive  process  in  the 
other. 

The  catabolic  activities  of  bacteria  con- 
sist in  the  fermentation  of  carbohy- 
drates and  in  the  cleavage  of  proteids 
and  fats  (see  ferments  or  enzymes; 
also  denitrifying  bacteria  in  nitrogen 
cycle). 

THE  ANABOLIC  OR  SYNTHETIC 
ACTIVITIES  OF  BACTERIA. 

The  depletion  of  the  soil  by  constant 
withdrawal  of  nitrogenous  substances 
by  plants  would  soon  be  complete, 
were  it  not  for  certain  forces  con- 
stantly at  work  replenishing  the  sup- 
ply from  the  free  nitrogen  of  the  air. 

Bacteria,  to  a  large  extent,  return  nitro- 
gen to  the  soil  (see  "Nitrogen  fix- 
ation," also  "Nitrification"  in  nitrogen 
cycle). 

^isrht  Production  by  bacteria,  is  seen  in 
certain  salt  water  forms.  Much  of 
the  phosphorescence  observed  at  sea 
is  caused  by  bacteria.  They  are  close- 
ly allied  to  the  putrefactive  bacteria 
and  in  the  sea  are  usually  found 
upon  rotting  animal  matter. 

The  light  production  is  dependent  upon 
free  access  of  oxygen  and  their  lum- 
inous quality  is  not  a  true  phospho- 
rescence in  that  it  does  not  depend 
upon  previous  illumination. 

The  formation  of  pigment  by  bacteria  is 
also  a  result  of  anabolic  activity. 

ANALYTIC  CHANGES  PRODUCED  BY 
MICRO-ORGANISMS. 

It  is  the  purpose  to  briefly  outline  the 
changes     induced,     with  particular 


18  BACTERIOLOGY. 


reference  to  the  so-called  cycles  of 
certain  of  the  elements  and  the  part 
played  by  microorganisms  in  inducing 
these  changes.     The  most  important 
elements  to  be  considered  are  nitrogen, 
carbon,  sulphur  and  phosphorus. 
1.  The  Cycle  of  Nitrogren  in  Nature. 
Nitrogen  is  found  in  nature  in  3  prin- 
cipal forms: — Free  in  the  air,  as  a 
gas;   in   inorganic  compounds  such 
as  ammonia,  nitrites,  and  nitrates; 
and  in  organic  compounds. 
Microorganisms     are     important  in 
changing  nitrogen  from  one  form  or 
combination    to    another;    in  fact, 
without  their  activity,  it  would  be 
impossible  for  higher  animals  and 
plants  to  exist  on  the  earth.    It  will 
probably    be    most    convenient  to 
start   with   complex   organic  nitro- 
genous compounds  in  the  study  of 
the  nitrogen  cycle  and  the  changes 
to  be  considered  are: 
(a)  Ammonification,    which    is  the 
conversion  of  complex  nitrogen- 
ous   compounds    into  simpler 
forms,  and  ultimately  into  am- 
monia. 

This  occurs  in  several  distinct 
stages.  The  complex  protein 
molecule  is  broken  up  into  some- 
what simpler  compounds — the 
»  proteoses,  and  then  into  pep- 
tones; (termed  peptonization). 
These  peptones  are  broken  down 
by  various  organisms  with  the 
formation  pf  polypeptids  and 
amino  acid's  primarily,  and  a 
considerable  number  of  second- 
ry  products. 

The  amino  acids  are  still  further 
decomposed  with  the  production 
of  ammonia. 

The  whole  process  of  successive 
cleavages  of  proteins  is  some- 
times termed  proteolysis.  The 
principal  nitrogenous  waste  pro- 
duct of  the  decomposition  of 
proteins  in  the  body  is  urea. 
This  is  actively  transformed  by 
certain  bacteria  into  ammonium 
carbonate.  It  is  evident  that 
all  organic  nitrogenous  com- 
pounds are  ultimately  reduced 
to  ammonia  by  the  process  of 
Ammonification. 


BACTERIOLOGY. 


19 


This  is  of  very  great  economic 
importance  in  agriculture,  as 
nitrogen  in  this  form  is  readily- 
changed  so  as  to  become  avail- 
able to  higher  plants. 

The  various  steps  in  ammonifica- 
tion  may  be  brought  about  by 
different  organisms.  A  few  spe- 
cies can  attack  native  proteins; 
many,  however,  can  utilize  and 
change  only  the  peptones,  the 
peptids  and  amino  acids.  The 
organisms  changing  urea  to  am- 
monium carbonate  constitute  a 
very  distinct  group. 

(b)  Nitrification.  (Nitrifying  Bac- 
teria). Certain  microorganisms, 
common  in  the  soil,  particularly 
a  coccus  (nitrosococcus),  are 
able  to  oxidize  ammonia  to 
nitrous  acid.  They  secure  their 
energy  for  growth  by  this 
change.  The  process  is  termed 
nitrosation.  Normally,  the  ni- 
trous acid  is  neutralized,  at 
once,  by  the  bases  of  the  soil 
thus  forming  nitrites.  These 
nitrites  soon  undergo  the  next 
change,  nitration,  or  oxidation 
to  nitrates  by  other  species  of 
soil  bacteria.  The  nitrates,  and 
to  a  less  degree  the  ammonia, 
constitute  the  source  of  nitro- 
gen for  higher  plants. 

No  nitrogenous  manure  is  effect- 
ive in  increasing  crop  yield  that 
is  not  capable  of  being  ammoni- 
fied and  nitrified. 

(c)  Nitrog-en  Assimilation  is  in 
large  part  a  function  of  the 
higher  plants. 

The  nitrates,  and  to  a  less  degree 
the  ammonia,  produced  by  bac- 
terial activity  in  the  soil  are 
taken  up  through  the  roots  and 
built  up  into  protoplasm  and 
complex  proteins. 

These  may  decaj^  or  they  may  be 
eaten  by  animals,  but  ultimate- 
ly they  are  decomposed  by  mi- 
croorganisms. 

This  alternate  synthesis  of  pro- 
teins by  higher  plants  and  dis- 
integration by  microorganisms 
constitutes  the  principal  part  of 
the  nitrogen  cycle. 


BACTERIOLOGY. 

(d)  Denltrlfication.  Nitrates,  in  the 
absence  of  oxygen  and  in  the 
presence  of  organic  matter,  may- 
reduce  to  nitrites  by  bacterial 
activity,  and  these  nitrites  fur- 
ther decompose  with  liberation 
of  free  nitrogen.  Under  these 
conditions,  microorganisms  take 
the  oxygen  from  the  molecule  of 
nitrate  or  nitrite.  Some  species, 
for  example,  will  live  under 
anaerobic  conditions  if  nitrates 
are  present,  otherwise  they  are 
aerobic. 

This  fact  is  of  some  significance 
in  agriculture  in  explaining  loss 
of  fertility  in  water-logged  soils. 

(e)  Kitrog'eii  Fixation.  If  gaseous 
nitrogen  is  lost  from  the  cycle 
as  a  result  of  denitrification, 
there  must  be  some  method 
whereby  it  can  be  again  fixed  or 
combined. 

A  certain  number  of  species 
among  the  bacteria  and  moulds 
are  known  to  possess  this 
power. 

Certain  of  the  higher  plants  have 
mould-like  fungi  which  live  up- 
on their  roots  and  take  up  the 
atmospheric  N  (e.  g.  Alders, 
Russian  olives,  and  certain 
other  trees,  the  orchids  and 
many  plants  living  in  peat  bogs 
and  swamps). 

These  organisms  are  termed  my- 
corrhizas. 

The  B.  radicicoliiB,  a  minute  bac- 
terium, produces  nodules  or  tu- 
bercles on  roots  of  many  legum- 
inous plants  as  the  bean,  pea, 
olive  and  alfalfa.  These  swell- 
ings are  found  to  be  made  up  of 
cells  tightly  packed  with  bac- 
teria. 

These  organisms  take  N  from  the 
air  and  directly  or  indirectly 
transfer  it  in  part  to  the  host 
plant.  Legumens,  unlike  most 
plants,  therefore,  can  grow  in 
soil  devoid  of  N.,  provided  the 
roots  are  supplied  with  nodules. 
The  efficiency  of  legumens  in  in- 
creasing the  fertility  of  the  soil 
is  due  to  this  fixation  of  nitro- 
gen. 


BACTERIOLOGY.  21 

There  are  also  a  few  living  soil 
bacteria  which  can  take  up 
nitrogen  from  the  air.  These 
belong  to  two  groups,  anaerobes 
and  aerobes;  some  spore  bearing 
soil  bacilli  (Clostridium),  in  the 
presence  of  proper  food,  such  as 
certain  carbohydrates,  can  fix 
some  nitrogen  under  anaerobic 
conditions.  These  forms  are  not 
very  important  in  the  soil. 

Much  more  important  are  the 
aerobic  Azotobacter.  These  se- 
cure energy  for  the  fixation  of 
nitrogen  by  the  oxidation  of 
carbohydrates.  They  are  prob- 
ably very  important  in  soil  fer- 
tility. 

2.  Carbon  Cycle.  This  cycle  in 
nature  as  affected  by  microorg- 
anisms is  more  simple  than  that 
of  N. 

It  is  well  here  to  emphasize  two 
facts: —  (1)  All  plants  and  an- 
imals alike  are  continuously 
developing  CO2;  (2)  Some  plants 
can  synthesize  organic  com- 
pounds, principally  carbohy- 
drates and  fats,  from  CO2. 

All  active  cells  are  constantly 
breaking  down  carbon  com- 
pounds; some  can  also  build 
them  up.  All  plants  containing 
chlorophyll  or  leaf-green  use 
CO2  and  water  to  produce  starch 
and  sugars,  gaining  the  energy 
necessary  by  means  of ,  the  ab- 
■  sorption  of  sunlight.  A  few 
bacteria  containing  bacterio- 
purpurin  are  also  capable  of 
using  light  for  this  purpose. 
Some  forms  oxidize  ammonia  i/) 
nitrites,  nitrites  to  nitrates, 
H2  S  to  sulphur  or  sulphur  to 
H2  SO4,  and  utilize  the  energy 
thus  secured  in  building  up  food 
materials. 

The  carbon  cycle,  then,  consists 
of  the  alternate  building  up  of 
carbon  into  organic  compounds 
and  their  subsequent  disintegra- 
tion with  ultimate  oxidation  of 
the  Carbon  to  CO2. 

3.  Stilphizr  Cycle.  The  decomposi- 
tion of  organic  compounds  con- 
taining sulphur  usually  results 
in  the  evolution  of  H2  S.  This 


22  BACTERIOLOGY. 


is  readily  oxidized  by  many 
aerobic  bacteria  with  the  pro- 
duction of  free  sulphur  and  sul- 
phuric acid.  These  organisms 
are  abundant  in  sewage  and  in 
water  of  sulphur  springs.  In 
these  springs,  they  may  form 
masses  of  considerable  size.  The 
sulphur  granules  may  be  seen 
within  the  cells  of  the  organism. 

Reduction  of  Sulphur  Compounds 
with  formation  of  H2  S  occurs 
when  sulphates  in  the  presence 
of  organic  matter  are  subjected 
to  anaerobic  conditions. 

The  sewage  of  some  cities  is  very 
offensive  because  the  city  water 
contains  sulphates  in  consider- 
able quantity.  Bacteria  cause 
decomposition  of  organic  mat- 
ter of  the  sewage  reducing  the 
sulphate  and  the  sulphite  is 
formed. 

Phosphorus  and  Calcium  Cycles 
in  nature  show  a  change  which 
is  influenced  by  microorganisms. 


CLASSIFICATION  OF  BACTERIA. 

Involution,  Structure,  Reproduction, 
Biologrical  Classification. 

Bacteria  are  minute  unicellular  or- 
ganisms which  may  occur  free  and 
singular,  or  in  larger  or  smaller  ag- 
gregations, thus  forming  multicellular 
groups  or  colonies,  the  individuals  of 
which  are,  however,  physiologically 
independent.  They  occupy  the  lowest 
plane  of  plant  life.  The  position  which 
they  occupy  in  plant  life  is  shown 
l>elow: — 

Plants. 

A.    Cryptogramia     (flowerless  plants 
forming  spores). 

1.  Fteridophyta.  e.  g.  ferns,  horse- 

tails and  club  mosses. 

2.  Bryophyta.  e.  g.  liverworts  and 

mosses. 

3.  Thallophyta.  e.  g. 

Myxomycetes  (slime-fungi). 

4.  Scliizophyta     (fission  plants) 

Schizophyceae   (fission  algae) 
Schizomycetes  (fission  fungi 
or  Bacteria. 


BACTERIOLOGY.  23 

Diatomea  (diatomis),  Chloro- 
phyceae  (green  algae). 

Rhodophycear  (red  algae), 
Phaeophyceae  (brown  algae). 

Characeae  (stone  worts),  Hy- 
pomycetes  (fungi). 

Lichens. 

B.    Fhanerog-amia^    (flowering  plants, 
forming  seeds). 


Bacteria,  Schizomycetes,  are  classi- 
fied by  Migula  into: — 
1.  Hubacteria,    cells    contain  no 

sulphur  granules  or  bacterio- 

purpurin. 

1.  Family    Coccaceae,  spherical 

forms 
Genus: 

(a)  Streptococcus,  non-motile; 

cells  divide  in  one  plane. 

(b)  Micrococcus,  non-motile, 

cells  divide  in  two  planes. 

(c)  Sarcina,     non-motile;  cells 

divide  in  three  planes. 

(d)  Planococcus,    motile;  cells 

divide  in  two  planes. 

(e)  Planosarcina,    motile;  cells 

divide  in  three  planes. 

2.  Family  Bacteriaceae,  straight, 

rod-shaped     forms  without 
envelope. 
Genus: 

(a)  Bacterium,  non-motile. 

(b)  Bacillus,     motile;  flagella 

over  whole  surface. 

(c)  Pseudomonas,  motile;  flagel- 

la polar. 

3.  Family   Spirillaceae,  curved 

rod-shaped     froms  without 
envelope. 
Genus: 

(a)  Spirosoma,  non-motile;  cells 

rigid. 

(b)  .  Microspira,     motile;  one,' 

rarely  two  or  three  polar 
flagella. 

(c)  Spirillum,    motile;  polar 

tufts  of  flagella. 

(d)  Spirochaeta,  cells  flexible. 

4.  Family  Chalamydobacteriaceae, 

cells  with  envelopes. 
Genus: 

(a)  Chlamydothrix,  unbranched 
threads;  cell-division  in 
one  plane;  (b)  crenothrix 
unbranched  threads;  cell 


24  BACTERIOLOGY. 


division  in  three  planes; 
sheath  visible. 

(c)  Phrag-midothrix,  unbranch- 

ed  threads;  cell-division 
in  three  planes;  sheath 
scarcely  visible. 

(d)  Sphoerotilus,  branched 

threads. 

II.  Thiobacteria,  cells  contain  sulphur 
granules  or  bacterio-purpurin;  red 
or  violet  color,  never  green. 

1.  Family  Beggiatoaceae,  thread- 

forming,    without  bacterio- 
purpurin. 
Genus: 

(a)  Thiotrix,  attached  threads; 

non-motile. 

(b)  Beggiatoa,  unattached 

threads;  motile. 

2.  Family  Rhodobacteriaceae,  cells 

contain  bacterio-purpurin 
and  sulphur  granules;  red  or 
violet. 

A.  Subfamily  Thiocapsaceae,  cells  di- 

vide in  three  planes. 
Genus:       Triocystis.  Thiocapsa. 
Thiosarcina. 

B.  Subfamily   Lamprocystaceae,  cells 

divide  first  in  three,  then  in  two 
planes. 

Genus:  Lamprocystis. 
Q.  Subfamily  Thiopediaceae,  cells  di- 
vide in  two  planes. 

Genus:  Thiopedia. 

D.  Subfamily  Amebobacteriaceae,  cells  ^ 

divide  in  one  plane. 
Genus:       Amebactor.  Thiothece. 
Thiodictyon.  Thiopolycoccus. 

E.  Subfamily  Chromatiaceae. 
Genus:    Chromatium.  Rhabdochro- 

matium.  Thiospirillum. 

A  commonly  used  classification  subdi- 
vides bacteria  into:* — 

I.  Xiower  Bacteria,  which  are  mi- 
croscopic in  size,  multiply  by 
fission  and  contain  no  chloro- 
phyll. 

1.  Cocci,  are  globular  in  form. 

(a)  Single  coccus. 

(b)  Diplococcus. 

(c)  Staphylococcus. 

(d)  Streptococcus. 

(e)  Tetrads. 

(f)  Sarcina. 

2.  Bacilli,  are  straight  rods, 
(a)  Long. 


BACTERIOLOGY. 


25 


(b)  Short. 

(c)  Diplobacillus. 

(d)  Irregular. 

3.  Spirillae  are  curved  or  spiral 
rods. 

(a)  Comma. 

(b)  Spiral. 

II.  Kig'lier  Bacterlar,  have  a  more  com- 
plex organization.  They  consist 
of  filaments  built  up  of  separate 
individuals,  some  of  which  seem 
related  to  physiologic  labor  and 
some  seem  for  the  purpose  of  re- 
production. 

They  possess  the  following  charac- 
teristics: They  are  attached, 
unbranched,  filamentous  forms, 
showing  a  differentiation  between 
base  and  apex;  growth  apparently 
apical;  exaggerated  pleomorphism; 
pseudo  branching  from  opposition 
of  cells  and  are  classified  into — 
Beggiota  and  thiothrix;  free  swim- 
ming forms,  which  contain  sul- 
phur granules. 

Crenothrix,  cladothrix  and  lepto- 
thrix  do  not  contain  sulphur  gran- 
ules. 

Streptothrix;  a  group  which  exhibits 
true  but  not  dichtomous  branch- 
ing and  contains  some  pathogenic 
species. 

Branched  forms  (normal  though  un- 
usual) must  not  be  confused  with 
involution  forms.  They  are  di- 
vided into: — 

1.  True  branching' — a  bud  springs 
out  from  the  bacteria,  e.  g.  Bacil- 
lus tuberculosis,  and  the  bacillus 
of  diphtheria. 

2.  Dichotomous,  which  is  often 
confounded  with  true  branching. 
It  is,  however,  a  misnomer,  as  it 
means  a  branching  in  two  equal 
parts. 

3.  Fseudodichotomous,  or  false 
branching  is  due  to  the  opposi- 
tion of  seperate  organism.  The 
streptococci  may  produce  false 
branching  by  one  cocci  dividing  at 
right  angles  to  chain  and  in  this 
way  producing  a  new  chain  of 
cocci  which  branches  from  the 
original  chain. 


26  BACTERIOLOGY. 

INVOLUTION  FORMS  OF  BACTERIA. 

Degreneratiou  Forms  or 
Fleomorphism. 

Bacteria  grown  on  artificial  media,  or 
having-  grown  in  the  same  media  for 
some  time;  i.  e.  under  conditions  not 
favorable  for  their  growth  may  show 
abnormal  or  unusual  shapes  (pleo- 
morphism). 

Involution  forms  characterized  by  alter- 
ations of  shape  are  not  necessarily 
dead,  but  those  forms  characterized  by 
a  loss  of  staining  power  are  always 
dead. 

STRUCTURE. 

Cell  membrane  or  Capsule  is  a  dense, 
highly  refractile,  gelatinous  outer 
portion  or  covering  of  the  cell  wall 
of  some  bacteria.  It  will  absorb 
moisture  and  swell.  Organisms  hav- 
ing a  capsule,  when  in  suitable 
solution,  make  the  solution  gelatin- 
ous or  slimy.  This  condition  gives 
rise  to  slimy  bread  and  ropy  milk. 
The  composition  of  the  capsule  may 
be  nitrogenous  or  non-nitrogenous. 
Substances  such  as  mucin,  mannans, 
.  galactans  and  dextrins  have  been 
identified. 

An  organism  may  produce  a  capsule 
•      under  certain  conditions  only,  as  in 
the  blood,  urine  or  milk,  but  not  in 
most  culture  media. 

The  membrane  prevents  certain  bac- 
teria, such  as  the  streptococcus  and 
the  staphylococcus,  from  becoming 
separated,  forming  them  into  chains 
or  bunches. 

Bacteria  growing  in  gelatinous  masses, 
secreted  by  the  cell,  is  known  as 
Zoogrloea.  It  can  be  seen  in  sewage 
and  on  filter  beds. 

The  capsule  is  not  easily  demonstrated 
by  the  ordinary  staining  methods. 
Cell  wall  lies  between  the  capsule  and 
the  cell  protoplasm,  from  which  it 
is  modified.  Its  chemical  composi- 
tion differs  in  different  bacteria. 

In  some  bacteria  it  is  of  a  cellulose 
reaction  in  others  an  albumin,  and 
in  chemical  composition  it  resembles 
the  chitin  of  the  lower  invertebrates. 

All   the   food  passes   through   it  by 


BACTERIOLOGY. 


27 


diffusion,  it  having  no  selective 
power.  It  can  be  easily  demon- 
strated. 

Cell  content  is  mainly  protoplasm,  com- 
posed of  mycoprotein.  As  a  rule  it 
is  homogeneous,  but  may  contain 
granules,  fluid  spaces,  fat  droplets, 
pigment,  sulphur  and  chlorophyll. 
That  portion  next  to  the  cell  wall, 
called  ectoplast,  is  an  important 
structure  as  it  has  to  do  with  nutri- 
tion. In  many  bacterial  cells  it  is 
semipermeable,  allowing  some  sub- 
stances to  pass  through,  inhibiting 
others.  A  demonstration  of  its  ac- 
tion as  an  osmotic  membrane  may 
be  had  by  placing  certain  bacteria  in 
strong  sugar  solutions,  causing  the 
protoplasm  to  shrink  (plasmolysis). 
A  definite  nucleus  has  not  been  demon- 
strated, though  the  granules  pres- 
ent are  probably  nuclear  in  nature. 
Their  behavior  during  cell  division 
would  probably  indicate  them  to  be 
a  primitive  nucleus. 

Metachromatic  graniaes  (e.  g.  diphthe- 
ria) derive  their  name  from  their 
ability  to  take  up  basic  aniline  dyes, 
as  does  chromatin. 

Sheath.  Certain  bacteria  growing  in  a 
chain  secrete  a  firm  membrane 
(sheath)  in  such  a  way  as  to  form 
a  tube  in  which  the  organism  lives, 
e.  g.  Chlamydothrix,  Crenothrix  and 
Cladothrix  substances,  such  as  iron 
or  calcium  compounds,  may  be  de- 
posited in  this  sheath. 

MOBILITY. 

1.  Mobility  by  Plaffella,  delicate  hair- 
like appendages  which  according  to 
some  investigators  are  out-growths 
of  the  cell  membrane,  or  of  the  cell 
wall  itself.  A  greater  majority, 
however,  believe  them  to  be  out- 
growths through  the  cell  membrane 
from  the  protoplasm.  They  are 
called: — 

(a)  Monotrichous,     when  situated 
singly  at  one  pole. 

(b)  Amphitrichous,    when  situated 
singly  at  each  pole. 

(c)  Lophotrichous,    when  situated 
plurally  at  each  pole. 

(d)  Peritrichous,     when  scattered 
around  the  entire  cell.  They 


28  BACTERIOLOGY. 

move  the  organism  fast  or  slow 
in  any  direction  away  from  its 
original  position  when  first  ob- 
served. 

They  are  very  difficult  to  demon- 
strate as  they  are  very  delicate, 
easily  break  off  and  disintegrate. 
Dark-field  illumination  and  spe- 
cial staining  methods  are  re- 
quired for  their  demonstration. 

2.  :Locomotion  by  undulatinsr  mem- 
branes has  been  observed  in  some 
bacteria. 

3.  Amoeboid  locomotion  has  been  found 
in  rare  instances. 

4.  Brownlan,  vibratory  or  molecular 
movement.  The  bacteria  vibrate, 
but  do  not  change  their  position. 
The  movement  is  due  to  transmis- 
sion by  external  physical  causes. 

REPRODUCTION. 

Under  this  head  is  considered  the:* — 

1.  Active  Stagre  (Vegetative),  i.  e.  by 
fission  or  simple  cell  division.  When 
conditions  such  as  heat,  moisture 
and  nutrition  are  favorable,  to- 
gether with  the  absence  of  the  dele- 
terious effects  of  other  bacteria,  or 

,  their  products 

(a)  The  cell  becomes  elongated  and 
the  protoplasm  aggregates  at 
opposite  poles. 

(b)  The  cell-wall  constricts,  usual- 
ly midway  between  the  proto- 
plasmic aggregations,  gradually 
forming  a  septum  in  the  interior 
of  the  cell. 

(c)  The  septum  divides  the  cell  into 
two  equal  parts. 

(d)  The  daughter  cells  may  remain 
united  by  the  gelatinous  en- 
velope for  a  variable  time. 
Eventually  they  separate  and 
they  themselves  subdivide. 

This  division  may  take  place  in 
one,  two  or  three  planes,  de- 
pending upon  the  nature  of  the 
organism. 

Division  may  be  completed  in  less 
than  30  minutes. 

2.  Resting  Stagre  (Spomlation). 
Spore    formation,     is  endogenous 
(Endosporous)      or  Arthrogenous 
(Arthrosporous). 


BACTERIOLOGY.  29 

Tike  requistes  for  spore  formation  were 
once  supposed  to  be: — 

(a)  An  exhaustion  of  nutriment. 

(b)  The  generation  within  the  me- 
dium of  toxic  material  from  the 
accumulation  of  metabolic  pro- 
ducts. 

(c)  The  environment  becomes  un- 
favorable, e.  g.,  temperature. 

In  other  words,  when  conditions 
became  such  that  the  cell  could 
no  longer  maintain  life,  the 
organism  turned  itself  into  a 
spore  in  order  that  it  might 
escape  annihilation. 

This  is  not  altogether  correct,  as 
sporulation  takes  place  only 
when  conditions  present  are 
most  favorable  to  the  well — 
being  of  the  cell.  The  tempera- 
ture at  which  spores  are  best 
formed  is  constant  for  each 
org^anism,  but  varies  with  the 
different  species,  aerobes  re- 
quire oxygen  for  sporulation  but 
anaerrobes  will  not  spore  in  its 
presence. 

Endogenous  spore  formation.  The  pro- 
toplasm of  the  cell  becomes  differ- 
entiated and  concentrates  into  a 
a  small  granule  which  increases  in 
size,  or  several  granules  are  formed, 
which  coalesce  and  grow  to  form 
an  oval  or  rarely  cylindrical  mass. 
Further  contraction  takes  place, 
the  outer  layers  become  still  more 
differentiated  and  form  a  distinct 
spore  membrane.  Some  authorities 
maintain  that  the  spore  membrane 
consists  of  two  layers,  the  ex- 
osporium  and  the  endosporium. 
The  spore  is  now  a  clearly  defined 

highly  refractile  body. 
The  cell  contains  but  one  spore,  situ- 
ated usually  in  the  middle,  occasion- 
ally at  one  end  (four  exceptions 
have  been  recorded,  e.  g.  B.  inflatus). 
It  is  of  the  same  diameter,  or  a  lit- 
tle less,  as  that  of  the  cell  itself. 

The  shape  of  the  parent  cell  may  be 
unaltered  (e.  g.  B.  anthrax)  or  al- 
tered (e.  g.  B.  tetanns),  and  this 
serves  for  a  classification  of  spore- 
bearing  bacilli:  viz: — 
(a)  Cell  body  unaltered  in  shape. 


30 


BACTERIOLOGY. 


(b)  Cell  body  altered  in  shape.  The 
terms  applied  to  each  are: — 

(1)  Clostridium.  (Spindle  shape). 
Swollen  at  the  center  and  thin 
at  the  poles. 

(2)  Cuneate.  (Wedge-shape). 

(3)  Clavate.  (Key-hole  shaped). 
Swollen  at  one  pole  and  unal- 
tered at  the  other. 

(4)  Capitate.    (Drum-stick  shaped). 
The  endospores  remain  within  the  cell 

for  a  variable  time,  but  are  eventu- 
ally set  free  by  the  swelling  up  and 
the  solution  of  the  cell  membrane  of 
the  parent  by  the  surrounding 
liquid  or  by  the  rupture  of  the  mem- 
brane. The  spore  now  presents  the 
following  characteristics: — 

(a)  Well  formed,  dense  cell  mem- 
branes, rendering  their  staining 
difficult;  and  when  stained, 
equally  difficult  to  decolorize. 

(b)  Highly  refractile,  which  differ- 
entiates it  from  vacuoles. 

(c)  Higher  resistance  (spore  re- 
sistance) than  the  parent  or- 
ganism on  account  of  the  low 
water  content  of  plasma,  low 
heat  conducting  power  and  the 
low  permeability  of  the  spore 
membrane  to  such  lethal  agents 
as  chemicals,  light,  heat,  desic- 
cation, starvation,  time,  etc., 
this  resistance  varying  some- 
what with  the  particular  spe- 
cies. 

Bacteria  grown  on  media  poor  in 
nutrient  material  are  likely  to  be- 
come asporogrenOTis ;  i.  e.,  they  be- 
come sterile  and  do  not  produce 
spores.  This  condition  may  be 
temporary  or  permanent. 

Arthrogrenous  spore  formation  is  seen 
only  in  the  micrococci.  One  com- 
plete element  resulting  from  fission 
becomes  differentiated  for  this  pur- 
pose, enlarges,  and  developes  a 
dense  cell  wall.  This  process  is 
probably  not  real  spore  formation 
but  a  relative  increase  in  resistance. 
They  have  never  been  seen  to  germ- 
inate, nor  is  their  resistance  very 
marked,  as  they  fail  at  culture  after 
having  been  exposed  to  80°  C  tem- 
perature for  10  minutes. 

Spore  Germination.  When  placed  under 
favorable  conditions  of  heat,  mois- 


BACTERIOLOGY.  51 

ture,  nutrition,  etc.,  the  spores 
germinate,  usually  within  24  to  36 
hours  and  successively  undergo  the 
following-  changes: — 

(1)  Swell  up  slowly  and  enlarge, 
through  absorption  of  water. 

(2)  Lose  their  refrangibility  (grow 
dull). 

(3)  One  of  the  following  processes 
is  observed  (a  particular  pro- 
cess is  constant  for  the  same 
species) : — 

(a)  The  spore  grows  out  into  the 
new  organism  without  throwing 
off  its  membrane. 

(b)  It  loses  its  spore  membrane  by 
solution. 

(c)  It  loses  its  spore  membrane  by 
rupture. 

(d)  Endo-germination.  The  spores 
germinate  within  the  parent 
body.  The  germinal  rod  be- 
comes detached,  leaving  the 
empty  capsule  within  the 
parent. 

The    rupture    may    be    polar  or 

equatorial. 
The  polar  rupture  may  take  place 

at   one   pole   only   or   at  both 

poles. 

In  the  cases  where  the  spore 
membrane  is  discarded,  the  cell 
membrane  of  the  new  bacillus 
may  be  formed  from: — 

(a)  The  inner  layer  of  spore  mem- 
brane, which  has  been  split  up 
into  a  parietal  and  visceral 
layer. 

(b)  The  outer  layers  of  the  cell 
protoplasm,  which  has  become 
differentiated  for  that  purpose. 

The  new  organism  now  increases 
in  size,  elongates  and  takes  on 
vegetative  growth. 
Foxrmatioxis  of  Gonidia.    In  the  higher 
bacteria   (filamentous  bacteria),  as 
in  Mycobacteriaceae,   a   number  of 
specialized  cells  or  spores  are  form- 
ed (short  rods  or  coccoid  forms)  by 
multiple    segmentation   or  differen- 
tiation, usually  at  the  free  tip  of 
the  filament,  and  are  termed  gonidia 
(conidia). 

They  may  be  termed  resting  bodies, 
as  they  remain  dormant  for  a  vari- 
able period  until  favorable  condi- 
tions are  brought  about,  when  they 


32  BACTERIOLOGY. 

elongate  and  produce  the  vegetative 
form  from  which  they  arose. 

Many  of  these  gonidia  have  been  con- 
sidered as  degenerative  forms,  but 
this  is  unlikely  as  degenerative 
elements  would  not  produce  new 
vegetative  cells.  According  to  A. 
Coffen  Jones,  tubercle  bacilli  pro- 
duce gonidia. 

The  resistance  of  the  diphtheria  or- 
ganism to  unfavorable  conditions 
would  make  it  likely  that  the  gran- 
ular segments  so  often  produced  are 
of  the  nature  of  gonidia. 

BIOLOGICAL  CLASSIFICATION. 

1.  Bacteria  are  classified  according  to 
their  life  functions  into: — 

(a)  Saprog-enic.  (Saprophytes),  or 
putrefactive  bacteria,  are  those 
that  live  only  on  dead  organic 
matter. 

(b)  Zymog'enlci,  f>r  fermentative 
bacteria,  are  those  which  pro- 
duce soluble  ferments  or  en- 
zymes during  the  course  of  their 
growth.  The  ferments  possess 
the  power  of  breaking  up  more 
highly  organized  nitrogenous 
and  non  nitrogenous  compounds 

»  into  simple  and  more  diffusible 

substances.  The  action  of  fer- 
ments upon  non-nitrogenous 
compounds  is  called  fermenta- 
tion. The  action  of  ferments 
upon  nitrogenous  compounds  is 
called  putrefaction,  often  pro- 
ducing odorous  gases  and 
ptomaines,  which  are  complex 
alkaloids  resembling  those 
found  in  plants. 
The  principal  bacterial  ferments 
are: — 

Proteolytic  (Converts  proteins 
into  proteose,  peptone  and 
further  products  of  hydrolysis). 

Diastase  (Converts  starches  into 
sugar). 

Znvertase  (Converts  saccharose 
into  a  mixture  of  dextrose  and 
levulose). 

Sennin  or  coagroIatiniT  (Coagu- 
lates milk  independent  of  the 
action  of  acids). 

(c)  Pathofifenic,  or  disease  produc- 
ing bacteria,  are  those  causing 


BACTERIOLOGY. 


33 


various  pathologrical  conditions 
and     producing-     the  diseases 
known  as  (infectious  diseases). 
Bacteria  are   classified  according  to 
their  food  requirements  into: — 

(a)  PrototropMc,  (e.  g.  nitrifying 
bacteria)  are  those  which  re- 
quire no  organic  food. 

They  change  albuminoids  into 
skatol,  indol,  leucin,  and  these 
into  nitrites  and  nitrites  into 
nitrates. 

(b)  Met  atrophic,  (e.  g.  saprophytes 
and  facultative  parasites),  are 
those  which  require  organic 
food. 

The  saprophytes  are  easily  culti- 
vated; some  will  grow  in  al- 
most pure  distilled  water  and 
some  will  grow  in  pure  solu- 
tions of  carbohydrates. 

The  facultative  parasites  need 
highly  organized  foods  as  pro- 
teids  or  other  sources  of  nitro- 
gen and  carbon  and  salts. 

(c)  Paratrophlc,  (e.  g.  obligate 
parasites)  are  those  which  re- 
quire living  food.  They  will 
not  live  outside  the  living  body. 

Bacteria  are  classified  according  to 
their  metabolic  products  into: — 
(a)  Chromogrenic,  or  pigrment-pro- 
ducing  bacteria,  are  those  which 
produce  vivid  pigments  (yellow, 
orange,  red,  violet,  fluorescent, 
etc.,)  during  the  course  of  their 
life  and  growth.  The  coloring 
matter  is  usually  an  intercell- 
ular excrementitious  substance; 
though  it  occassionally  appears 
to  be  stored  within  the  body  of 
the  organism.  They  are  there- 
fore classified  into: — 
Chromoparous  bacteria,  when  the 
pigment  is  diffused  out  upon 
and  into  the  surrounding 
medium. 

Chromophorous  bacteria,  when  the 
pigment  is  stored  in  the  cell 
protoplasm  of  the  organism. 

Parachromophorous  bacteria, when 
the  pigment  is  stored  in  the  cell 
wall  of  the  organism. 

Different  species  of  chromogenic 
bacteria  differ  in  their  require- 
ments as  to  environment  for  the 
production  of  their  character- 


34 


BACTERIOLOGY. 


istic  pigrments;  some  need  oxy- 
gren,  light  or  high  temperatures; 
others  favor  the  opposite  condi- 
tions. 

(b)  Photogrenlc,  or  light-producing 
bacteria,  are  those  which  exhibit 
phosphorescence  when  culti- 
vated under  suitable  conditions. 

(c)  Aerog'enlc,  or  gas  producing 
bacteria,  are  those  which  pro- 
duce hydrogen,  carbon  dioxide 
and  sulphuretted  hydrogen,  etc. 

Toxins.  Many  bacteria,  especially  the 
pathogenic,  elaborate  or  secrete 
poisonous  substances,  concerning 
which  little  exact  knowledge  is 
available,  though  many  appear  to 
be  enzymic  in  their  action.  They 
seem  to  be  akin  to  the  venom  of 
serpents  and  other  animals  and  to 
certain  poisonous  principles  of 
plants. 

It  has  been  estimated  that  1-1000  gm. 
of  tetanus  toxin  will  kill  a  horse 
weighing  1,200  pounds.  They  were 
first  called  ptomaines  or  cadaveric 
alkaloids,  but  this  term  is  now  ap- 
plied to  poisons  which  form  in  de- 
composing meat,  cheese,  etc.,  as  a 
result  of  chemical  change  caused  by 
bacteria;  they  have  also  been  termed 
>  toxalbumlns,  as  they  give  all  the 
reactions  of  albumin.  It  is  probable, 
however,  that  a  toxalbumin  is  but 
a  combination  of  the  toxin  and  the 
substances  derived  from  the  me- 
dium of  growth. 

A  certain  group  of  toxins  are  retained 
within  the  organism  and  are  only 
set  free  after  its  death. 

Toxins  are  usually  divided  into: — 

Xntracellnlar  (inseparate)  are  those 
which  are  bound  up  with  the  pro- 
toplasm of  the  organism. 

No  means  has  as  yet  been  devised  for 
their  separation  of  extraction.  Anti- 
bacterial seram  is  used  to  combat 
this  type. 

Sxtracellnlar  (soluble)  are  excreted 
by  the  organism  and  are  diffused 
into  and  held  in  solution  by  the  sur- 
rounding  medium. 

Anti-toxin  serum  is  used  to  combat 
this  type. 

End-products  of  metabolism  are  or- 
ganic acids  (lactic,  butyric,  pro- 
pionic, benzoic,  formic,  acetic,  oxalic. 


BACTERIOLOGY. 


35 


succinic,  salicylic,  gallic  and  tan- 
nic), alkalies  (ammonia),  aromatic 
compounds  (indol,  phenol),  reducing 
substances  (nitrates  to  nitrites), 
and  gases  (sulphuretted  hydrogen, 
carbon  dioxide,  etc.) 
Growth.  Certain  conditions  are  neces- 
sary to  the  life  and  growth  of 
bacteria;  any  marked  change  in 
these  conditions  will  inhibit  the 
growth  or  destroy  them.  Water  is 
absolutely  essential  for  their  growth. 

1.  Influence  of  atmosphere.  Certain 
bacteria  require  oxygen  for  their 
growth  and  death  will  follow  if  this 
is  not  available.  They  are  termed 
olillg'ate  aerobes. 

A  certain  group  of  bacteria  will  thrive 
equally  well  with  or  without  oxygen. 
They  are  termed  facultative  anae- 
robes. 

Certain  bacteria  live  and  multiply 
only  when  there  is  complete  exclu- 
sion of  free  oxygen.  They  are 
termed  obllgrate  anaerobes. 

2.  Influence  of  heat.  A  temperature 
of  from  10''  to  40°  C  is  necessary 
to  the  life  and  growth  of  bacteria. 
Practically  no  growth  occurs  below 
^°  C,  and  very  little  above  40°  C. 
The  most  favorable  temperature  for 
the  majority  of  microorganisms  is 
from  30°  C  to  37°  C.  Saprophytes 
grow  between  0°  and  30°  C,  the 
optimum  being  15°  to  20°  C. 

Parasites,  flourish  between  10°  and  45° 
but  best  at  body  temperature,  37°  C. 

The  maximum  and  minimum  temper- 
atures at  which  growth  takes  place, 
as  well  as  the  optimum,  are  fairly 
constant  for  each  bacterium.  They 
may  be  classified,  according  to  their 
optimum  temperature,  into: — 


MIN.   OPT.  MAX. 

(a)  Psychrophilic 
(chiefly  water 

organisms)  .  .    0°  C.  15°  C.  30°  C. 

(b)  Mesophi  1  1  c 
(includes 
pathogenic 

forms)   15°  C.  27°  C.  45°  C. 

(c)  Thermophylic 

bacteria   45°  C.  55°  C.  70°  C. 

Each  bacterium  has  its  own  Thermal 
death  point. 


36  BACTERIOLOGY. 

The  "thermal  death  point"  of  an  or- 
ganism is  that  temperature  which 
causes  the  death  of  the  vegetative 
forms  when  the  exposure  is  con- 
tinued for  a  period  of  10  minutes. 

It  is  between  50°  and  60°  C  in  the 
most  pathogenic,  while  below  the 
lower  limit  their  growth  is  only 
inhibited.  An  exposure  to  250°  C. 
has  been  made  without  preventing 
the  organisms  future  development. 

Spores  are  extremely  resistant;  some 
are  killed  only  after  an  hour's  ex- 
posure to  115°  C. 

3.  Influence  of  ligrlit.  Many  organisms 
are  indifferent  to  the  presence  of 
light.    On  the  other  hand 

Daylight  frequently  inhibits  the 
growth  and  alters  to  a  greater  or 
lesser  extent  the  biochemical  char- 
acters of  the  organisms;  e.  g., 
chromogenicity  or  power  of  lique- 
faction. Pathogenic  bacteria  under- 
go a  progressive  loss  of  virulence 
when  cultivated  in  the  presence  of 
daylight. 

Direct  sunlight  destroys  them  as  does 
also  electric  light,  but  to  a  less  ex- 
tent. Violets  rays  are  very  effective 
in  the  destruction  of  bacteria. 
.4.  Influence  of  electricity.  Electrical 
currents  inhibit  or  destroy  the 
growth  of  bacteria,  not  directly, 
but  probably  by  the  products  of 
electrolysis. 

Roentgen  rays  are  bactericidal  to 
bacteria  in  living  tissues,  but  have 
little  effect  on  cultures. 

5.  Influence  of  movements.  Movements, 
if  slight  and  of  a  flowing  character, 
do  not  seem  to  affect  the  growth  of 
bacteria,  but  violent  shaking  kills 
them. 

CULTIVATION  OF  BACTERIA. 

Culture  Media,  Tubing*  Media, 
Sterilization. 

As  it  is  difficult  and  sometimes  impos- 
sible to  study  the  growth  of  bacteria 
m  their  natural  habitat,  it  becomes 
necessary  to  isolate  individual  mem- 
bers of  microorganisms,  to  observe 
their  growth,  morphology,  phenomena, 
etc.,  by  their  cultivation  on  artificial 
nutrient  naedia. 


BACTERIOLOGY. 


37 


APPARATUS  BEQUIBEB. 

Test  tubes.  Several  sizes  should  be 
kept  in  stock.  The  ordinary  tubes 
in  most  use  are  the  %"x5"  Board 
of  Health  tubes.  Small  tubes  5x0.9 
cm.  for  use  in  inverted  position  in- 
side tubes  containing-  carbohydrate 
media  as  gas  collecting  tubes. 

Plorence  Plasks  of  250,500  and  1000 
cc.  capacity  will  be  found  very  con- 
venient. 

Erlenmeyer  Plasks,  with  narrow  neck 
of  75,  100,  150  and  250  cc.  capacity. 

Petri  Dishes  or  Plates,  1.5  cm.  high  X 
10  cm.  diameter  put  up  in  bundles 
of  6,  wrapped  in  paper  or  cloth, 
sterilized  and  put  aside  for  use. 
They  can  also  be  put  up  in  specially 
prepared  metallic  boxes. 
Pipettes  of  1  cc.  plain,  1  cc.  gradu- 
ated in  0.  1  cc,  and  .01  cc.  capacity; 
also  pipettes  of  10  cc.  capacity 
g-raduated  in  .1  cc.  Each  variety 
should  be  stored  in  large  test  tubes 
or  in  special  metallic  boxes,  steril- 
ized and  put  aside  for  use. 

Capillary  pipettes  (Pasteur's)  are  pre- 
pared from  small  bore  soft  glass 
tubing,  heated  and  pulled  out  to  a 
fine  capillary  tube  at  one  end. 

Blood  pipettes  (Pakes)  are  made,  from 
1  cm.  bore  soft  glass  tubing-,  in  a 
manner  similar  to  Pasteurs,  except 
that  they  are  pulled  out  at  both 
ends.  Wright's  tubes  are  similar 
to  Pakes'  except  that  one  end  is 
turned  at  an  angle.  They  are  stored 
in  test  tubes,  sterilized  and  put 
aside  for  use. 

Permentation  tubes,  used  for  the  col- 
lection and  analysis  of  gases  liber- 
ated from  media  during  the  growth 
of  some  bacteria.  They  may  be 
plain  or  graduated.  They  are 
plugged  with  cotton  and  sterilized. 

Platinum  wire,  fitted  into  a  glass  or 
aluminum  handle,  to  be  used  for 
inoculations. 


CULTURE  MEDIA. 

The  greater  number  of  these  media  are 
primarily  fluid,  but  in  order  to  bet- 
ter study  the  characteristics  of  in- 
dividual organisms,  through  their 
colonies  many  media  are  therefore 


BACTERIOLOGY. 


rendered  solid  by  the  addition  of 
substances  like  grelatin  or  agar  in 
varying  proportions.  Gelatin  is 
employed  for  the  solidification  of 
those  media  on  which  it  is  intended 
to  cultivate  bacteria  at  room  tem- 
perature or  in  the  "cold"  incubator. 
Gelatin,  in  the  precentage  usually 
employed,  becomes  liquid  at  25°  C. 

Agar,  in  the  precentage  usually  em- 
ployed, only  becomes  liquid  when 
exposed  to  90°  C  for  a  considerable 
period  and  again  solidifies  at  40°  C. 

Such  media  is  spoken  of  a  liquefiable 
media.  Other  media  as  potato,  co- 
agulated blood  serum,  etc.,  can  not 
be  again  liquefied  and  are  therefore 
spoken  of  as  solid  media. 

Meat  Extract  forms  the  basis  of  sev- 
eral of  the  nutrient  media  and  is 
prepared  as  follows: — 

1.  Add  to  1000  cc.  distilled  water,  in 

an  enameled  pot,  500  gms.  of 
finely  minced  fresh  lean  meat. 

2.  Heat  gently  in  a  water  bath,  at  a 

temperature  that  at  no  time  ex- 
ceeds 40°  C,  for  20  minutes;  this 
will  dissolve  out  the  soluble  pro- 
teids,   extractives,   salts,  etc. 

3.  Raise  the  temperature  of  the  mix- 

ture to  boiling  and  maintain  for 
10  minutes;  this  precipitates 
some  of  the  albumins,  haemog- 
lobin, etc.,  from  the  solution. 

4.  Strain    through    muslin  (sterile) 

or  a  perforated  porcelain  fun- 
nel, then  filter  through  paper 
into  a  sterile  flask  and  when 
cool  make  up  loss  by  evapora- 
tion to  1000  cc.  with  distilled 
water. 

5.  If   not   needed  at   once,  sterilize 

for  20  minutes  on  3  consecutive 
days. 

Wyeth's    beef-juice,    or  Liebig's 
extract  of  meat,  3  gms.  to  1000 
cc.  of  distilled  water  heated  and 
filtered  as  above,  may  be  sub- 
stituted, except  where  the  more 
highly  parasitic  bacteria  are  to 
be  cultivated. 
The  Reaction  of  Meat  Extract  as  pre- 
pared above  is  always  acid,  due  to 
acid  phosphates   of  potassium  and 
sodium,  acids  of  the  glycollic  series, 
and  acid  organic  compounds. 


BACTERIOLOGY. 


39 


Prolonged  boiling  causes  the  extract 
to  undergo  hydrolytic  changes 
which  increase  its  acidity.  It 
should  therefore  be  boiled  for  at 
least  45  minutes,  when  it  will  be- 
come stable,  and  the  total  acidity  is 
to  be  estimated  when  the  solution 
>v    is  at  the  boiling  point. 

The  meat  extract  reacts  acid  to 
phenolphthalein,  though  it  may  re- 
act neutral  or  alkaline  to  litmus, 
due  to 

(1)  The  insensitiveness   to  some  or- 

ganic acids. 

(2)  The  formation  of  dibasic  sodium 

phosphate,    formed   during  the 
process  of  neutralization. 
STANDARDIZING  THE  REACTION. 

1.  Fill  a  burette  with  standardized 

n/20  NaOH. 

2.  Measure  out  5  cc.  of  media  and 

45  cc.  distilled  water  into  a 
beaker  (should  be  at  a  temper- 
ature of  100°  C). 

3.  Add  to  the  contents  of  the  beaker, 

5  drops  of  a  0.5%  (50%  alcohol) 
solution  of  phenolphthalein. 

4.  From  the  burette,   run   the  n/20 

NaOH  solution  carefully  into 
the  test  media,  constantly  stir- 
ring until  the  end-point  is 
reached,  as  indicated  by  a  deep- 
rose  color. 

5.  Read   ofC   the   amount   of  NaOH 

solution  required  to  neutralize 
the  5  cc.  of  media. 

6.  Verify   the   reaction   by  another 

titration. 

7.  Calculate  the  amount  of  standard- 

ized normal  NaOH  it  will  take 
to  neutralize  the  remaining  990 
cc.  of  media. 
(For  all  practical  purposes  it  can 
be  estimated  as  still  having 
1000  cc.  of  media  and  adding  the 
normal  NaOH,  viz: — if  it  re- 
quires 5  cc.  of  the  n/20  NaOH 
to  neutralize  5  cc.  of  the  media, 
then  50  cc.  of  the  normal  NaOH 
will  be  required  to  neutralize 
the  1000  cc.  of  media.  In  other 
words,  move  the  decimal  point 
one  to  the  right,  e.  g.,  burette 
reading  is  5.3  cc,  then  53.  cc. 
will  neutralize  the  1000  cc.  of 
media).  The  sign  +  (plus)  is 
prefixed  to  the  media  if  it  is 


40 


BACTERIOLOGY. 


acid  and  the  sign  —  (minus) 
if  it  is  alkaline,  e.  g.,  media  + 
10  indicates  that  it  reacts  acid 
to  phenolphthalein  and  would 
require  the  addition  of  10  cc. 
normal  NaOH  per  100  cc.  for 
neutralization. 
8.  Titrate  again  the  neutralized 
media  to  insure  results.  In  as 
much  as  the  titration  for  a  last 
control  is  often  wanted,  it  may 
be  well  to  use  a  deka-normal 
NaOH  for  the  neutralization  of 
the  bulk,  so  as  not  to  bring  the 
total  quantity  of  media  greatly 
above  the  original  1000  cc,  as 
might  be  the  case  if  the  N/20 
or  normal  NaOH  were  used. 
Nearly  all  bacteria  have  a  well 
marked  "optimum  reaction" 
which  happily  approximates 
close  to  +  10,  therefore  this 
standard  may  be  used  for  all 
media  unless  otherwise  indi- 
cated. 

The  standardizing  1000  cc.  of 
media  to  +  10  is  accomplished 
by  merely  subtracting  10  of  the 
NaOH  from  the  initial  calcu- 
lation. This  renders  the  reac- 
tion -f  10. 


FILTRATION  OF  MEDIA. 

Flnld  Media  are  filtered  through  filter 
paper  folded  in  the  "physiological- 
filter  form  so  as  to  accelerate  the 
rate  of  filtration. 

^Iquefiable  Media  are  filtered  through 
"paper  Chardin,"  which  is  a  special- 
ly made  filter  paper. 
Gelatin   if  made  properly   will  filter 

through  this  paper  readily. 
Agar,  likewise,  if  properly  made  will 
filter  readily,  but  not  so  rapidly  as 
gelatin. 

A  special  hot-water  jacket  has  been 
constructed  to  surround  the  glass 
funnel;  the  temperature  of  the 
water  in  the  jacket  is  maintained 
at  90°  C.  and  facilitates  the  filtra- 
tion. If  care  is  taken  the  liquefiable 
media  can  be  filtered  through  ab- 
sorbent cotton  efficiently. 


BACTERIOLOGY. 


41 


STOCK  MEDIA. 

Bouillon.  Put  500  cc.  double  strength 
meat  extract  into  a  litre  flask  and 
add  300  cc.  distilled  water. 
Mix  10  gms.  peptone  and  5  gms,  salt 
into  a  smooth  paste  with  200  cc.  of 
distilled  water  previously  heated  to 
60°  C.  Add  the  emulsion  to  the 
meat  extract  and  heat  in  the  Arnold 
for  45  minutes  to  dissolve  the  pep- 
tone and  to  render  the  acidity  of 
the  meat  extract  stable.  Estimate 
the  reaction  and  control  the  results. 
Heat  agrain  in  Arnold  for  30  minutes 
to  completely  precipitate  the  phos- 
phates. Filter  through  paper  into 
flask.  Sterilize,  or  tube  and  ster- 
ilize. 

Ag'ar=Ag'ar.  (Agar  is  derived  from  sea 
plants  along  the  coast  of  Japan.  It 
has  some  of  the  properties  of 
gelatin,  but  is  less  affected  by  heat). 
Weigh  a  2  litre  double  Agate  ware 
boiler  and  note  it.  Put  500  cc.  dou- 
ble strength  meat  extract  into  the 
boiler.  Mix  10  gms.  of  peptone, 
5  gms.  of  salt  into  a  paste  with 
150  cc.  distilled  water.  Add  the 
paste  and  15  to  20  gms.  of  Agar 
(powdered  if  available)  to  the  meat 
extract.  Heat  over  flame  to  100°  C. 
for  25  minutes  (stirring  constantly) 
or  more  for  complete  solution  of 
Agar.  Weigh  the  pan  and  to  Its 
contents  add  enough  water  to  make 
up  the  bulk  of  the  medium  to  1 
litre. 

Titrate,  control  the  result,  calculate 
the  amount  of  soda  solution  re- 
quired to  make  the  medium  +  10 
and  add  it  to  the  medium.  Place  in 
the  Arnold  or  over  the  flame  for  20 
minutes  to  complete  the  precipita- 
tion of  the  phosphates,  etc. 

Cool  the  medium  to  60°  C,  add  the 
whipped  white  of  two  eggs,  place 
it  over  the  gas  burner  or  in  Arnold 
until  the  egg-albumin  has  formed 
into  a  firm  floating  mass.  Filter 
through  paper,  tube  and  sterilize. 
Gelatiji  is  used  for  determining  the  pro- 
teolytic ferments  of  bacteria  by  its 
liquefaction.  Other  distinctions  ar# 
also  met  with. 


42 


BACTERIOLOGY. 


Weigrh  a  2  litre  double  Agate  boiler 
and  note  it.  Put  500  cc.  double 
strength  meat  extract  into  the 
boiler.  Mix  10  gms.  of  peptone,  5 
gms.  of  salt  into  a  paste  with  150 
cc.  distilled  water.  Add  the  paste 
and  100  to  150  gms.  sheet  gelatin 
(cut  into  small  pieces)  to  the  meat 
extract.  Heat  over  flame  to  100°  C. 
for  10  minutes  (stirring  constantly 
till  there  is  complete  solution  of  the 
gelatine.  Weigh  the  pan  and  its 
contents  and  add  enough  water  to 
make  up  the  bulk  of  the  medium  to 
1  litre.  Titrate,  control  the  result, 
calculate  the  amount  soda  solution 
required  to  make  the  medium  +  10 
and  add  it  to  the  medium.  Place 
in  the  Arnold  or  over  the  flame  for 
20  minutes  to  complete  the  precipi- 
tation of  the  phosphates,  etc.  Cool 
the  medium  to  60°  C,  add  the 
whipped  white  of  two  eggs,  place 
it  over  the  flame  or  in  the  Arnold 
until  the  egg  albumin  has  formed 
into  a  flrm  floating  mass.  Filter 
through  paper,  tube  and  sterilize. 

Blood  Serum.  The  blood  is  collected 
at  the  slaughter  house  in  sterile 
glass  cylinders  and  allowed  to  stand 
for  15  minutes  to  form  clot  to  pre- 
vent the  serum  from  being  stained 
with  haemoglobin.  When  removed 
to  the  laboratory  the  clot  is  separ- 
ated from  the  sides  of  the  cylinder 
by  a  sterile  glass  rod  and  placed  in 
the  ice  chest  for  24  hours.  The 
serum  is  then  drawn  out  with 
sterile  pipettes  and  placed  in  sterile 
test  tubes  (5  cc.  in  each).  The 
tubed  serum  is  heated  on  two  suc- 
cessive days.  The  third  day, 
heat  the  tubes  in  a  slanting  posi- 
tion in  a  serum  inspissator  to 
about  72°  C.  which  coagulates  the 
serum.  Place  the  tubes  in  the  in- 
cubator at  37°  C  for  48  hours  to 
eliminate  the  tubes  that  have  been 
contaminated.  Store  in  a  cool  place. 
The  serum  can  be  sterilized  by  the 
fractional  method  by  exposure  in  a 
water  bath  to  a  temperature  of  56* 
C.  for  30  minutes  on  each  of  6  con- 
secutive days.  Store  in  the  fluid 
condition  and  coagulate  in  the  in- 
spissator when  needed. 


BACTERIOLOGY.  43 

Guy's  Citrated  Blood  Agrar.  A  small 
rabbit  is  killed  by  chloroform, 
nailed  out  on  a  board,  hair  moisten- 
ed thoroug-hly  with  2%  solution  of 
lysol.  skin  is  reflected  (with  sterile 
instruments)  over  the  thorax,  thorax 
opened  (sterile),  pericardium  opened 
(sterile),  surface  of  left  ventricle 
seared  with  hot  iron,  the  point  of  a 
sterile  capillary  pipette  is  thrust 
through  the  wall  of  the  ventricle, 
pipette  filled  with  blood  by  suction, 
transfer  the  blood  to  a  small 
Erlenmeyer  flask  containing  a  num- 
ber of  glass  beads  and  5  cc.  con- 
centrated sodium  citrate  solution, 
agitate,  set  aside  for  2  hours,  with 
a  sterile  10  cc.  graduated  pipette, 
transfer  1  cc.  citrated  blood  to  a 
tube  of  liquefied  agar,  mix,  allow 
agar  to  cool  in  slanting  position, 
place  tubes  in  incubator  for  48 
hours,  after  which  time  store  the 
uncontaminated  tubes  for  future 
use. 

Potato.  Cylinders  are  cut  out  of  a  well 
washed  peeled  potato.  The  cyl- 
inders are  cut  obliquely  from  end  to 
end,  forming  them  into  wedges. 
The  fresh  potato  is  strongly  acid 
and  in  order  to  approximate  +  10 
the  cylinders  are  placed  in  1%  solu- 
tion of  sodium  carbonate  for  30 
minutes.  Each  wedge  is  placed  in 
a  test  tube  into  which  has  been  pre- 
viously inserted  a  piece  of  ab- 
sorbent cotton  moistened  with 
sterile  water,  with  its  base  resting 
upon  the  cotton.  The  tubes  are 
then  replugged  and  sterilized  in 
Arnold  on  each  of  3  consecutive 
days. 

The  acid  of  the  potato  can  also  be 
abstracted  by  placing  the  wedges  in 
running  water  for  24  hours. 

Dorset's  Effgr.  Sterilize  in  the  autoclave 
for  20  minutes  1  litre  of  a  .85% 
solution  of  sodium  chloride  and  cool 
to  20^  C.  Wash  12  fresh  eggs  with 
water,  then  with  pure  formaline  and 
allow  them  to  dry.  Break  the  eggs 
into  a  sterile  graduate,  noting  their 
total  volume.  Add  the  salt  solution 
to  the  eggs  in  proportion  of  1  to  3. 
Whip  the  mixture  with  an  egg- 
whisk  thoroughly  and  filter  through 


44 


BACTERIOLOGY. 


coarse  muslin  into  a  sterile  flask. 
(A  few  drops  of  alcoholic  solution 
of  basic  fuchsin  to  Rive  a  definite 
pink  color,  or  a  few  drops  of  water 
proof  Chinese  ink  added  to  the 
medium  at  this  stag-e  will  facilitate 
the  subsequent  "fishing  of  col- 
onies"). 

Tube  and  solidify  at  a  slant  in  the 
inspissator  at  75°  C.  for  one  hour. 
Incubate  for  48  hours  and  eliminate 
the  contaminated  tubes.  The  sterile 
tubes  are  capped  with  rubber  caps 
and  stored  for  future  use, 

Hgg.  A  number  of  eggs  are  broken  into 
a  vessel  and  thoroughly  mixed  with 
a  little  water,  tubed  and  sterilized 
at  a  slant  in  the  Arnold  on  each  of 
3  consecutive  days. 

Dunham's  Peptone.  10  gms.  of  peptone 
and  5  gms.  of  salt  are  emulsified 
with  250  cc.  of  distilled  water  pre- 
viously heated  to  60°  C.  The 
emulsion  is  placed  in  a  flask  and 
made  up  to  1  litre  with  distilled 
water.  Heat  in  Arnold  for  30 
minutes,  filter  through  paper,  tube 
and  sterilize  in  Arnold. 

Dextrose  Bouillon.  Make  bouillon  in  the 
manner  outlined  above  and  add  to 
•  it  1%  of  dextrose.  Tube  and  ster- 
ilize as  for  bouillon.  This  media 
is  generally  used  in  the  fermenta- 
tion tubes.  The  ordinary  glucose 
will  answer  as  well  except  that 
during  its  sterilization  it  will 
deepen  greatly  in  color. 

Milk.  1  litre  of  fresh  milk  is  put  into- 
a  large  separating  funnel  and  heat- 
ed in  the  Arnold  for  1  hour.  Esti- 
mate the  reaction.  (If  it  is  higher 
than  +  20  or  lower  than  -f  10  re- 
ject it.)  Cool  to  separate  the  fat. 
Draw  off  the  fat-free  milk  into 
sterile  tubes  and  sterilize  in  the 
Arnold  for  20  minutes  on  each  of  5 
successive  days.  Incubate  for  48 
hours  and  eliminate  any  contam- 
inated tubes. 

Xiitmus  Milk.  The  milk  is  prepared  as 
described  above,  and  fat-free  is 
drawn  off  into  a  sterile  flask.  Suf- 
ficient sterile  litmus  solution  is 
added  to  give  it  a  deep  lavender 
color.    Tube  and  sterilize  as  above. 


BACTERIOLOGY.  45 


SPECIAL  MEDIA. 

ANAEROBIC  CUXiTUBES. 

Kltasato's  Glucose  Formate  Bonlllon. 

Dissolve  20  gms.  of  glucose  and  4 
g-ms.  of  sodium  formate  in  1  litre 
bouillon.  Tube  and  sterilize  in 
Arnold. 

Weyle's  Sulphlndlsrotate  Bouillon.  Dis- 
solve 20  gms.  glucose  and  1  gm.  of 
sodium  sulphindigotate  in  1  litre 
bouillon.  Tube  and  sterilize  in 
Arnold. 

Kitasato's  Glucose  Formate  Gelatine. 

Dissolve  20  gms.  of  glucose  and  4 
gms.  of  sodium  formate  in  1  litre 
of  hot  gelatin.  Filter  through 
paper,  tube  and  sterilize  in  Arnold. 

Weyl's  Sulphlndlgrotate  Gelatin.  Dis- 
solve 20  gms.  glucose  and  1  gm.  of 
sodium  sulphindigotate  in  1  litre 
hot  gelatine.  Filter  through  paper, 
tube  and  sterilize  in  Arnold. 

Xltasato's  Glucose  Formate  Agrar. 
Dissolve  29  gms.  glucose  and  4  gms. 
sodium  formate  in  1  litre  hot  agar. 
Tube  and  sterilize  in  Arnold. 

Sulphlndlgrotate  Agar.  Dissolve  20 
gms.  glucose  and  1  gm.  sodium 
sulphindigotate  in  1  litre  hot  agar. 
Tube  and  sterilize  in  Arnold. 

All  the  sulphindigotate  media  are  of 
a  blue  color.  During  the  growth  of 
the  anaerobes,  the  media  is  oxidized 
and  changed  in  color  to  a  light 
yellow. 

MacConkey's  Bile  Salt  Broth.  Emul- 
sify 20  gms.  of  peptone  in  200  cc. 
distilled  water  previously  warmed 
to  60°  C.  Dissolve  5  gms.  sodium 
taurocholate  and  5  gms.  of  glucose 
in  the  emulsion.  Wash  the  emul- 
sion into  a  flask  with  800  cc.  of  dis- 
tilled water  and  place  in  Arnold 
for  20  minutes  at  lOO**  C.  Filter 
through  paper  and  add  sterile 
litmus  solution  until  the  medium 
is  of  a  deep  purple  color.  Tube  into 
gas  tubes  and  sterilize  in  Arnold 
for  20  minutes  on  3  consecutive 
days. 

FOB  THE   STUDY  OF  THB  ORGAN- 
ISM'S CKEMICAi;  COMPOSITION'. 
Uschlnsky's  Asparagine.    Dissolve  8.4 
gms.  asparagine,  10  gms.  ammonium 
lactate,  5  gms.  sodium  chloride,  0.2 


46  BACTERIOLOGY. 


gms.  magnesium  sulphate,  0.1  gm. 
calcium  chloride  and  1  gm.  acid 
potassium  phosphate,  in  1  litre  of 
distilled  water.  Add  40  cc.  glycerine, 
tube  and  sterilize  in  Arnold. 

This  media  can  be  made  up  into 
gelatine  or  agar. 

Usclxiiuiky's  Proteid  Free  Broth.  Dis- 
solve 0.1  gm.  calcium  chloride,  0.2 
gms.  magnesium  sulphate,  2  gms. 
acid  potassium  phosphate,  3  gms. 
potassium  aspartate,  5  gms.  sodium 
chloride  and  6  gms.  ammonium  lac- 
tate, in  1  litre  of  distilled  water. 
Add  30  cc.  glycerine,  tube  and  steril- 
ize. 

FOB  THE  STUDY  OF  THE  ORGAN- 
ISM'S BIO-CHEMICAl^  REACTION. 
Bnnhatn'g  Inosite-free  Bouillon.  In- 
oculate 1  litre  of  bouillon  with  the 
B.  lactis  aerogenes  and  incubate  for 
48  hours.  Heat  in  Arnold  for  20 
minutes.  Estimate  the  reaction  and 
make  it  +  10.  Inoculate  with  the 
B.  coli  communis  and  incubate  for 
48  hours.  Heat  in  Arnold  for  20 
minutes. 

Fill  2  fermentation  tubes,  tint  with 
litmus  solution  and  sterilize;  in- 
oculate with  the  B.  lactis  aerogenes. 
If  no  acid  or  gas  is  formed  the 
medium  is  sugar  free;  but  if  acid  or 
gas  is  present,  again  make  the 
bouillon  to  +  10  reinoculate  with 
either  of  the  above  bacteria  and  in- 
cubate; make  another  test.  Repeat 
above  procedure  till  neither  acid  or 
gas  appears. 

Stand  the  medium  in  a  cool  place  to 

allow  the  growth  to  sediment. 
Filter  the  top  medium  through 
paper  till  clear.  Tube  and  sterilize 
in  the  Arnold. 
Nitrate  Bouillon.  Dissolve  5  gms.  of 
potassium  nitrate  in  1  litre 
bouillon.  Tube  and  sterilize  in 
Arnold. 

Iiitmus  Bouillon.  Add  enough  sterile 
litmus  solution  to  1  litre  of  bouillon, 
to  give  it  a  dark  lavender  color. 
Tube  and  sterilize  in  Arnold,  (+10 
media  will  usually  react  faintly 
alkaline  or  occasionally  neutral  to 
litmus). 


BACTERIOLOGY.  47 

Iron  Bouillon.  Dissolve  1  gm.  of 
ferric  tartrate  in  1  litre  bouillon. 
Tube  and  sterilize  in  Arnold. 

£ead  Bouillon.  Dissolve  1  grm.  of 
lead  acetate  ,  in  1  litre  bouillon. 
Tube  and  sterilize  in  Arnold. 

Fake's  Nitrate  Peptone.  Emulsify  10 
gms.  peptone  with  200  cc.  ammonia 
— free  distilled  water  previously 
heated  to  60*  C.  Wash  emulsion 
into  a  flask  and  make  up  to  1  litre 
with  ammonia-free  distilled  water. 
Heat  in  Arnold  for  20  minutes. 
Dissolve  1  gm.  of  sodium  nitrate  in 
the  above.  Filter  through  paper, 
tube  and  sterilize. 

Rosalie  Acid  Peptone.  Make  a  .5%, 
80%  alcoholic,  solution,  of  rosalic 
acid  (corallin)  for  a  stock  solution. 
Add  to  100  cc.  Dunham's  peptone, 
2  cc.  of  the  corallin  stock  solution. 
Heat  in  Arnold  for  30  minutes. 
Filter  through  paper,  tube  and 
sterilize. 

Pakes'  Iron  Peptone.  Emulsify  30 
gms.  of  peptone  with  200  cc.  tap 
water  (heated  to  about  60"  C.) 
Wash  it  into  a  flask  with  800  cc.  of 
tap  water.  Dissolve  in  it  5  gms. 
of  salt  and  3  gms.  of  sodium  phos- 
phate. Heat  in  Arnold  for  30  min- 
utes. Filter  and  tube.  Add  to  each 
tube  0.1  cc.  of  a  2%  neutral  solu- 
tion of  ferric  tartrate.  Sterilize. 

l^ead  Peptone.  Prepare  as  for  iron 
peptone  except  to  substitute  0.1  cc. 
of  a  1%  neutral  aqueous  solution 
of  lead  acetate  for  the  ferric  tar- 
trate. 

Capaldi-Proskauer  No.  1.  Dissolve 
2  gms.  sodium  chloride,  0.1  gm. 
magnesium  sulphate,  0.2  gms.  cal- 
cium chloride  and  2  gms.  mono- 
potassium  phosphate  in  1  litre  of 
distilled  water.  Add  to  the  mixture, 
2  gms.  of  asparagin  and  2  gms.  of 
mannite.  Take  25  cc.  of  mixture 
and  titrate  it  against  n/10  sodium 
hydrate  using  litmus  as  an  indi- 
cator. Calculate  amount  of  sodium 
hydrate  necessary  to  make  the  so- 
lution neutral  to  litmus  and  add 
it.  Filter  and  add  to  it  5%  of 
neutral  litmus  solution.  Tube  and 
sterilize  in  Arnold. 

Capaldi-Proskauer  No.  2.  Dissolve  20 
gms.  of  peptone  and  1  gm.  of  man- 


48  BACTERIOLOGY. 

nite  to  1  litre  of  distilled  water. 
Neutralize  as  In  No.  1,  filter  and 
add  litmus  solution  as  above.  Tube 
and  sterilize  in  Arnold. 

Glucose  Gelatine.  Dissolve  20  gms. 
of  glucose  in  1  litre  of  hot  gelatin, 
filter  through  paper,  tube  and  steril- 
ize in  Arnold. 

Glucose  Asrar.  Dissolve  20  gms.  of 
glucose  in  1  litre  of  hot  agar,  filter, 
tube  and  sterilize  in  Arnold. 

Urine  Gelatine.  Fresh  urine  with  a 
sp.  gr.,  of  1010  (if  above  1010,  it  is 
diluted  with  sterile  water  until  that 
sp.  gr.,  is  reached)  is  collected  in  a 
sterile  fiask,  heated  to  the  boiling 
point  and  the  reaction  estimated 
and  noted.  10%  of  gelatine  is  added 
and  the  mixture  heated  in  the 
Arnold  for  1  hour.  Estimate  the 
reaction  to  that  of  the  original 
urine.  Cool  to  60"  C.  and  clear  with 
egg.  Filter  through  paper,  tube  and 
sterilize  in  Arnold. 

Heller's  Urine  Gelatin.  Same  as  above 
with  addition  of  1%  of  peptone  and 
0.5%  of  salt. 

Urine  Agfar.  To  fresh  urine  with  a 
specific  gravity  of  1010  is  added  1.5 
,  to  2%  of  powdered  agar.  Heat  in 
the  Arnold  for  1  and  %  hours.  Cool 
to  60**  C.  and  clear  with  eggs. 
Filter,  tube  and  sterilize  in  Arnold. 

Eiss'  Serum  Dextrose.  Blood  is  col- 
lected and  allowed  to  clot.  It  is 
then  expressed  in  a  graduated  cyl- 
inder and  for  every  100  cc.  of  serum 
300  cc.  of  distilled  water  is  added. 
Heat  in  the  Arnold  for  30  minutes. 
Filter  if  turbid.  (If  not  needed  at 
once  it  can  be  sterilized  for  3  days 
and  stored). 

Dissolve  10  gms.  of  dextrose  in  1 
litre  of  the  above  serum.  Filter 
through  paper  and  add  50  cc.  of  the 
"neutral  litmus  solution."  Tube  and 
sterilize  in  Arnold. 


FOR  THE  STUDY  OF  CHBOMOGENZC 
ORGANISMS. 

Eisenbergr's  MUk  Rice.     Mix   70  cc. 

bouillon  and  210  cc.  milk.  In  a 
mortar  rub  up  100  gms.  of  rice 
powder  and  the  mixture  into  a 
paste.  Spread  the  paste  out  in  0.5 
cm.  thick  layer  over  the  bottom  of 


BACTERIOLOGY. 


49 


petri-dishes.  With  the  lids  removed, 
heat  over  a  water  bath  at  100*  C. 
until  the  mixture  solidifies. 
Replace  the  lids  and  sterilize  in  the 
Arnold. 

FOB  THE  STUDY  OF  FKOSPKOBES- 
CENT  AND  FKOTOGESriC  OR- 
GANISMS. 
Fish  Bouillon.  Dissolve  26.5  gms. 
sodium  chloride,  0.75  gm.  potassium 
chloride  and  3.25  gms.  magnesium 
chloride  in  500  cc.  distilled  water. 
Add  the  solution  to  500  gms.  of 
herring,  cod  or  mackerel  (in  an 
enameled  pot)  and  heat  over  a 
water  bath,  gently  at  40^*  C.  for  20 
minutes,  then  rapidly  raise  to  100" 
C,  maintaining  it  at  this  tempera- 
ture for  10  minutes.  Strain  through 
muslin.  Emulsify  5  gms.  of  pep- 
tone in  200  cc.  of  fish  water,  then 
mix  it  thoroughly  with  the  rest. 
Heat  in  the  Arnold  for  20  minutes. 
Filter  through  paper  and  when  cold 
make  up  to  1  litre  with  distilled 
water.  Tube  and  sterilze  in  Arnold. 
If  it  is  to  be  used  as  a  basis  for 
gelatin  or  agar  it  should  be  made 
up  double  strength.  The  gelatine 
or  agar  is  then  prepared  in  the 
ordinary  manner. 

FOB  THB   STUDY  OF  BABTH  BAC- 

TBBIA. 

Idpnian  and  Brown's  Bairthy  Salts 
Agfar.  (Enumeration  of  soil  or- 
ganism). Emulsify  20  gms.  agar 
with  200  cc.  distilled  water  and 
wash  it  by  means  of  400  cc.  of  dis- 
tilled water  into  a  double  boiler. 
Add  to  it  an  emulsion  made  with 
.5  gm.  of  peptone  and  50  cc.  of  dis- 
tilled water  and  heat  at  100°  C.  for 
20  minutes.  Add  to  the  mixture, 
10  gms.  of  dextrose,  0.5  gm.  potas- 
sium phosphate,  0-2  gm.  magnesium 
sulphate  and  0.06  gm.  of  potassium 
nitrate.  Adjust  the  weight  of  the 
mass  to  the  calculated  figure  for 
1  litre  (1025  gms.)  by  adding  dis- 
tilled water  at  100°  C.  Titrate  and 
adjust  the  reactif)n  to  -|-  5.  Cool  to 
60°  C,  clear  with  egg,  filter,  tube 
and  sterilize,  in  Arnold. 

Beyrinck's  No.  1.  (Cultivation  of  ni- 
trogen fixing  organisms).  Dissolve 
1    gm.    potassium    hydrogen  phos- 


50  BACTERIOLOGY. 

phate,  0.2  gm.  magrnesium  sulphate 
and  0.02  gm.  sodium  chloride  in  1 
litre  of  water.  Add  1  cc.  of  a  1% 
solution  of  manganese  sulphate. 
Add  20  gms.  dextrose  and  heat  in 
Arnold  for  20  minutes.  Filter,  tube 
and  sterilize. 

Beyrinck's  No.  2.  (For  growth  of 
Azobacter).  It  is  the  same  as 
No.  1,  except  that  mannite  is  sub- 
stituted for  dextrose. 

Winogradsky's  for  Nitric  Orgranisms. 
Dissolve  1  gm.  potassium  phosphate, 
0.5  gm.  magnesium  sulphate,  0.01 
gm.  calcium  chloride  and  2  gms. 
sodium  chloride  in  1  litre  of  dis- 
tilled water.  Fill  into  flasks  in 
quantities  of  20  cc.  and  add  to  each 
a  small  amount  of  freshly  washed 
magnesium  carbonate.  Sterilize  in 
Arnold  as  usual.  Add  to  each  flask 
2  cc.  of  a  sterile  2%  solution  of 
Ammonium  sulphate.  Incubate  and 
eliminate  any  flask  not  sterile. 

Winogradsky's  for  Nitrous  Organisms. 
Dissolve  1  gm.  ammonium  sulphate 
and  1  gm.  of  potassium  sulphate  in 
1  litre  of  distilled  water.  Add  5  to 
10  gms.  basic  magnesium  carbonate 
,  previously  sterilized  by  boiling. 
Fill  into  flasks,  etc.,  as  for  the  nitric 
organisms. 

rOB   THE    STUBY    OF    WATER  OB- 
OANISMS. 

Hesse  and  Keyden  Naehrstoff  Agar. 

(For  enumeration  of  organisms) 
Emulsify  12.5  gms.  of  agar  in  250 
cc.  of  distilled  water.  Wash  the 
emulsion  into  a  double  boiler  with 
250  cc.  of  distilled  water.  Heat  in 
water  bath  till  agar  is  dissolved 
and  add  to  it  an  emulsion  made 
from  7.5  gms.  NaehrstofC-Heyden 
(albumose)  with  200  cc.  cold  dis- 
tilled water. 
Adjust  weight  of  mass  (1020  gms.) 
to  1  litre  by  adding  distilled  water 
at  100°  C.  Clear  with  eggs.  Tube 
and  sterilize  in  Arnold. 

FOB    THE    STUDir    OF    FI^ANT  OR- 
GANISMS. 

Haricot  Bouillon,  (For  bacteria  from 
tubercles  of  legumens).  Add  to  1 
litre  of  distilled  water,  250  gms. 
of  haricot  beans,   10  gms.  nodium 


BACTERIOLOGY. 


51 


chloride  and  1  cc.  of  a  1%  solution 
of  sodium  bicarbonate.  Heat  in 
Arnold  for  30  minutes.  Filter  and 
add  20  gms.  saccharose.  Tube  and 
sterilize. 

Haricot  Affar.  In  the  usual  way  add 
15  gms.  of  agar  to  haricot  bouillon, 
adjust  the  weight  and  reaction,  cool 
to  60°  C,  clear  with  eggs.  Filter, 
add  the  20  gms.  saccharose,  tube 
and  sterilize. 

Hay  Infusion.  Macerate  10  gms.  of 
chopped  hay  with  1  litre  of  distilled 
water  that  has  been  heated  to  70°  C. 
in  a  flask;  close  flask  with  rubber 
stopper  and  place  in  a  water  bath 
at  60°  for  3  hours.  Replace  the 
stopper  with  a  cotton  plug  and  heat 
in  Arnold  for  1  hour.  Filter  through 
paper,  tube  and  sterilize. 

Beets,  Carrots,  Turnips  and  Parsnips 
are    prepared    in    the    manner  de- 
scribed for  potato. 
FOR  STUDY  COIiI-TYFHOID  GROUP 
OP  OSaANISMS. 

Carbolized  Bouillon.  Dissolve  1  gm. 
of  carbolic  acid  in  1  litre  of 
bouillon.  (2.5  to  5  gms.  are  also 
used.)   Tube  and  sterilize  in  Arnold. 

Carbolized  Gelatin.  Dissolve  5  gms. 
of  carbolic  acid  in  1  litre  of  hot 
nutrient  gelatin.  Tube  and  sterilize 
in  Arnold. 

Carbolized  Agrar.  Dissolve  1  gm.  of 
carbolic  acid  in  1  litre  of  hot  nu- 
trient agar.  Tube  and  sterilize  in 
Arnold. 

Parietti's  Bouillon.  Mix  4  cc.  of  pure 
hydrochloric  acid  with  100  cc.  of  a 
5%  carbolic  acid  solution  and  allow 
to  stand  for  a  few  days.  Prepare 
several  nutrient  bouillon  tubes,  each 
containing  10  cc,  sterilize,  add  to 
each  the  above  solution  by  means 
of  a  sterile  pipette  in  quantities  of 
0.1,  0.2  and  0.3  cc.  Incubate  for 
48  hours  to  eliminate  the  contam- 
inated tubes. 

Litmus  Gelatin.  Add  to  nutrient 
gelatin  enough  sterile  neutral  litmus 
to  give  it  a  deep  lavender  color. 
Tube  and  sterilize  in  Arnold. 

Litmus  Lactose  Bouillon.  Emulsify  4 
gms.  of  peptone  with  200  cc.  of  meat 
extract  at  60°  C.  Mix  2  gms.  of 
salt  and  20  gms.  of  lactose  with 
the  emulsion. 


52  BACTERIOLOGY. 

Add  to  the  mixture  200  cc.  of  meat 
extract  and  600  cc.  of  distilled  water 
and  heat  in  Arnold  for  30  minutes. 

Neutralize  carefully  to  litmus  paper. 
Heat  in  Arnold  for  20  minutes. 
Filter  through  paper  and  add  sterile 
litmus  solution  to  color  medium  a 
deep  purple.  Tube  and  sterilize  in 
Arnold. 

Wurtz's     ZiltmuB     ]&actose  Gelatin. 

Render  1  litre  nutrient  gelatin  —  5. 
Heat  in  Arnold  for  20  minutes. 
Clear  with  egg.  Dissolve  20  gms. 
lactose  in  the  medium.  Filter  and 
add  enough  litmus  solution  to  color 
medium  a  pale  lavender.  Tube  and 
sterilize  in  Arnold. 

Wurte's  liltmus  Iiactose  Agfar.  Render 
1  litre  nutrient  agar  —  5.  Heat  in 
Arnold  for  20  minutes.  Cool  to  60° 
C.  and  clear  with  egg.  Dissolve  20 
gms.  lactose  solution  to  color 
medium.  Filter  and  add  enough 
litmus  solution  to  color  medium  a 
pale  lavender.  Tube  and  sterilize 
in  Arnold. 

MacConkey's  BUe  Salt  Agrar.  Emul- 
sify 15  gms.  powdered  agar  with 
200  cc.  tap  water  at  60"  C.  Mix  the 
emulsions.  Dissolve  5  gms.  sodium 
»  taurocholate  in  300  cc.  tap  water 
and  with  it  wash  the  emulsions 
into  a  double  boiler.  Heat  in  water 
bath  or  Arnold  for  20  minutes.  Ad- 
just the  weight  of  the  medium  mass 
for  1  litre  (1040  gms.)  Cool  to 
60°  C.  and  clear  with  eggs.  Filter 
and  add  10  gms.  lactose.  Tube  and 
sterilize  in  Arnold. 

Fawcus'  Bile  Salt  Agrar.  Emulsify 
20  gms.  of  agar  with  100  cc.  of  dis- 
tilled water.  Wash  the  emulsion 
into  a  double  boiler  with  500  cc.  of 
distilled  water.  Heat  until  the  agar 
Is  dissolved.  Cool  to  60"*  C.  and 
clear  with  eggs.  Filter  and  add  5 
gms.  of  sodium  taurocholate,  20 
gms,  of  peptone  and  5  gms.  of 
lactose.  Adjust  reaction  to  +  15. 
Filter,  add  20  cc.  of  a  1%  aqueous 
solution  of  picric  acid.  Tube  and 
sterilize  In  Arnold. 

Glycerine  Potato  Bonillon.  Grate  1 
kilo  of  potatoes  previously  well 
washed  and  peeled.  Weigh  and  add 
distilled  water  in  proportion  of  1  cc. 
for  every  gm.  of  potato.    Place  in 


BACTERIOLOGY. 


61 


ice  chest  for  12  hours.  Strain  and 
filter  through  paper.  Note  amount. 
Add  an  equal  quantity  of  distilled 
water.  Heat  in  Arnold  for  60 
minutes.  Add  4%  glycerine.  Mix 
and  filter.    Tube  and  sterilize. 

XSlsuer's  Potato  Gelatine.  Grate  1  kilo 
of  potatoes  previously  well  washed 
and  peeled.  Weigh  and  add  dis- 
tilled water  in  proportion  of  1  cc. 
for  every  gm.  of  potato.  Place  in 
ice  chest  for  12  hours.  Strain  and 
filter  through  paper.  Note  the 
amount.  Add  15%  of  gelatin  and 
place  in  Arnold  till  dissolved.  Esti- 
mate the  reaction  and  adjust  it  to 
4-  25.  Cool  to  60°  C.  and  clear  with 
eggs.  Add  1%  powdered  potassium 
iodide.  Filter  through  paper,  tube 
and  sterilize  in  Arnold. 

Braiim's  Puchsin  Ag'ar.  Prepare  a 
fuchsin  solution  as  follows: —  Dis- 
solve 3  gms.  basic  fuchsin  in  60  cc. 
absolute  alcohol.  Put  aside  for  24 
hours,  then  centrifugalize  thorough- 
ly and  decant  the  top  fluid  and  place 
in  a  well-stoppered  bottle. 

Dissolve  10  gms.  lactose  in  1  litre 
nutrient  agar.  Adjust  the  reaction 
to  —  5.  Filter  and  thoroughly  mix 
with  5  cc.  of  fuchsin  solution.  Add 
to  the  mixture  25  cc.  of  a  freshly 
prepared  10%  aqueous  solution 
sodium  sulphite.  Tube  and  sterilize. 
Store  in  a  dark  place. 
POB  THE  STUDY  OP  MIIiK  OBGAK- 
ISMS. 

Iiitmus  Whey.  Curdle  fresh  milk  by 
adding  rennet  and  warming  to  60" 
C.  Filter  off  the  whey  and  neu- 
tralize to  litmus  by  adding  a  40% 
solution  of  citric  acid.  Heat  in 
Arnold  for  one  hour  to  coagulate 
all  the  proteids.  If  the  whey  is 
cloudy,  put  it  aside  in  ice-chest  for 
48  hours,  decant  and  filter  into  a 
sterile  flask.  Add  litmus  solution 
till  the  whey  is  of  a  deep  purple 
red  color.    Tube  and  sterilize. 

Petruschky's  Litmus  Whey.  Add  1.5 
cc.  of  hydrochloric  or  glacial  acetic 
acid  to  1  litre  fresh  milk.  Filter 
off  the  casein  and  neutralize  to 
litmus  by  adding  normal  sodium  hy- 
drate solution.  Boil  and  neutralize 
to  litmus  by  adding  n/10  sodium 
hydrate  solution.     Filter  and  add 


54  BACTERIOLOGY. 

litmus  solution  till  the  whey  is  of 
a  deep  purple  color.  Tube  and 
sterilize. 

Whey  Gelatin.  Curdle  fresh  milk  by 
adding  rennet  and  warming  to  60** 
C.  Filter  and  estimate  the  reaction. 
Add  10%  gelatine  and  place  in 
Arnold  till  dissolved.  Weigh  and 
estimate  the  reaction  of  the  mass. 
Restore  the  mass  to  the  original  re- 
action of  the  whey  by  sodium  hy- 
drate solution.  Cool  to  60°  C.  and 
clear  with  eggs.  Filter,  tube  and 
sterilize  in  Arnold. 

iitmus  Whey  Agar  or  Gelatin.  Add 
1.5  cc.  of  hydrochloric  or  glacial 
acetic  acid  to  1  litre  fresh  milk  and 
boil  for  5  minutes.  Filter  and 
render  whey  faintly  alkiline  to 
litmus.  Prepare  an  emulsion  from 
a  few  cc.  of  the  whey  and  10  gms. 
of  peptone.  Add  the  emulsion  to 
the  whey.  Mix  in  50  gms.  gelatin 
(or  15  gms.  agar)  and  heat  in  the 
Arnold  till  dissolved.  Clear  with 
eggs,  filter  and  adjust  the  weight 
of  the  medium  mass  for  1  litre.  Add 
15  gms.  dextrose.  Color  with 
sterile  litmus  solution.  Tube  and 
sterilize  in  Arnold  on  each  of  5 
.consecutive  days. 

Gelatin  Agfar.  Emulsify  5  gms.  powd- 
ered agar  with  100  cc.  of  distilled 
water.  Add  to  it  400  cc.  of  double 
strength  meat  extract  and  100  gms. 
of  gelatin.  Heat  in  the  Arnold  till 
dissolved.  Emulsify  10  gms.  of 
peptone  and  5  gms.  salt  with  100  cc. 
double  strength  meat  extract  heated 
to  60°  C.  and  add  it  to  the  medium 
mixture.  Heat  in  the  Arnold  for 
15  minutes,  adjust  the  weight  of 
the  medium  mass  for  1  litre  by 
adding  distilled  water  at  100°  C, 
estimate  the  reaction  and  adjust  it 
to  +  10;  heat  in  Arnold  for  20 
minutes,  cool  to  60°  C,  clear  with 
eggs,  filter  through  paper,  tube  and 
sterilize  in  Arnold.  (This  medium 
will  allow  incubation  at  a  tempera- 
ture above  22  C.  If  incubation  at 
30°  C.  is  to  be  employed,  use  10% 
of  gelatin  and  0.5%  of  agar  in  the 
medium.  If  incubation  at  37°  C.  is 
used,  make  the  medium  with  12% 
gelatin  and  0.75%  agar.  Avoid  the 
addition  of  too  much  agar,  as  the 


BACTERIOLOGY. 


55 


liquefying  ferment  may  be  retard- 
ed or  absent). 
rOR  THE  STUDY  OF  DIPI.OCOCCUS 
PNEUMONIA. 

Washbouru's  Blood  Agfar.  Incubate 
ag-ar  slants  for  48  hours  to  evapor- 
ate ofC  some  of  the  water  of  con- 
densation; under  aseptic  conditions 
open  the  thorax  of  a  small  rabbit 
and  with  sterile  pipettes  deliver 
from  the  heart  a  small  quantity  of 
blood  over  the  surface  of  each  of 
the  agar  slants;  allow  the  blood  to 
coagulate  in  a  slanted  position;  in- 
cubate the  blood  agar  for  48  hours 
and  eliminate  any  contaminated 
tube. 

FOB  THE  STUDY  OF  DIFI^OCOCCUS 
MENINGITIDIS  INTRACEI^IiU- 
I.ARIS. 

Wassenuann  Ascitic  Agrar.     Add  to 

210  cc.  of  distilled  water  90  cc  of 
ascitic  fluid  and  6  gms.  of  nutrose. 
Heat  over  a  flame,  with  constant 
-shaking,  until  the  fluid  boils  and  the 
nutrose  is  dissolved.  Add  the  mix- 
ture to  600  cc.  of  melted  nutrient 
agar,  heat  in  the  Arnold  for  30 
minutes,  filter,  tube  and  sterilize. 

FOR  THE  STUDY  OF  QONOCOCCUS. 

I^ipschuetz's  Egrg*  Albumin  Broth.  In 

a  flask  containing  some  sterile  glass 
beads  place  4  gms.  of  powdered  egg 
albumin  and  200  cc.  of  distilled 
water  previously  warmed  to  37**  C. 
Dissolve  the  egg-albumin  by  shak- 
ing. Add  40cc.  of  n/10  NaOH. 
Allow  to  stand  for  30  minutes  with 
frequent  shaking.  Filter,  sterilize 
by  boiling  two  or  three  times  at 
intervals  of  two  hours;  add  600  cc. 
nutrient  bouillon,  fill  in  quantities 
of  50  cc.  into  small  fiasks,  incubate 
for  48  hours  and  eliminate  any 
contaminated  flask. 
Egrgr  Albumin  Agfar.  This  is  prepared 
in  the  same  manner  as  the  above 
except  that  nutrient  agar  is  sub- 
stituted for  the  600  cc.  of  nutrient 
bouillon. 

Serum  Bouillon.  Under  aseptic  pre- 
cautions, hydrocele,  pleuritic  or 
ascitic  fluid  is  collected  in  sterile 
flasks;  add  to  it  twice  its  bulk  of 
sterile  nutrient  bouillon;  if  neces- 
sary filter;  tube,  sterilize  in  water 


56  BACTERIOLOGY. 


bath  at  56°  C.  for  30  minutes  on 
each  of  5  consecutive  days,  incubate 
for  48  hours  and  eliminate  any  con- 
taminated tubes. 
Wertlieimer's  Serum  Agar.  Prepare 
nutrient  agar  using  2%  of  agar,  2% 
peptone,  5%  salt  and  q.  s.  meat  ex- 
tract. Adjust  the  reaction  to  +  10, 
filter,  tube  in  quantities  of  5  cc.  and 
sterilize.  After  last  sterilization 
cool  to  42°  C.  and  add  5  cc.  sterile 
blood  serum  from  human  placenta 
to  each  tube,  slope  the  tubes;  in- 
cubate, when  solid,  for  48  hours 
and  eliminate  any  contaminated 
tubes. 

Heiman's  Semm  Affar.  Prepare  nu- 
trient agar  using  2%  of  agar,  1.5% 
peptone,  0.5%  salt  and  q.  s.  meat 
extract.  Adjust  the  reaction  to  + 
10,  filter,  tube  in  quantities  of  cc. 
and  sterilize.  After  the  last  sterili- 
zation cool  to  42°  C.  and  add  3  cc. 
sterile  hydrocele  fluid,  ascitic  fluid 
or  pleuritic  effusion,  to  each  tube; 
slope  the  tubes;  incubate,  when 
solid,  for  48  hours  and  eliminate 
any  contaminated  tubes. 

FOB  THE  STUDY  OF  B.  DIPHTHERIA. 

I^oeffler's  Blood  Serum.  Prepare  nu- 
trient bouillon  using  veal  meat  ex- 
tract instead  of  beef.  Add  to  the 
bouillon  1%  of  glucose  and  when 
dissolved  add  300  cc.  of  clear  blood 
serum  to  every  100  cc.  of  bouillon. 
Fill  into  a  sterile  tube  and  complete 
as  for  blood  serum. 

Councilman  and  Mallory's  Blood 
Serum.  Collect  blood  in  slaughter- 
house, coagulate,  remove  the  serum 
and  tube  (avoid  air  bubbles).  Heat 
the  tubes  at  a  slant  in  the  hot  air 
sterilizer  at  90°  C.  till  coagulated 
(%  hour),  then  sterilize  in  Arnold 
for  20  minutes  on  each  of  3  succes- 
sive days. 

FOR  THE   STUDY  OF  B.  TUBERCU- 
I.OSIS. 

Glycerine   Bouillon.     Add   60   cc.  of 

glycerine  to  1  litre  of  nutrient 
bouillon.  Tube  and  sterilize  in 
Arnold. 

Glycerine  Agrar.  Add  60  cc.  of  glyc- 
erine to  1  litre  of  nutrient  agar. 
Tube  and  sterilize  in  Arnold. 

Glycerine  Blood  Senuu.    Add  5%  of 


BACTERIOLOGY. 


57 


glycerine  to  blood  serum  before 
tubing",  then  proceed  as  described 
under  Blood  Serum. 
Glycerinated  Potato.  Prepare  the 
ordinary  potato  slants  and  soak 
them  in  25%  solution  of  glycerine 
for  15  minutes.  Moisten  the  cotton 
pads  at  the  bottom  of  the  tube  with 
a  25%  solution  of  glycerine.  Tube 
and  sterilize  in  Arnold  for  20 
minutes  on  each  of  5  consecutive 
days. 

NEUTBAZi  UTMUS  SOXiUTION. 

Place  50  gm.  of  litmus  in  300  cc.  95% 
alcohol  and  put  it  aside  until  al- 
cohol acquires  a  green  color  (com- 
pleted in  about  7  days  with  daily 
shaking).  Decant  oft  the  green 
alcohol  and  again  treat  with  300  cc. 
95%  alcohol  and  repeat  shaking. 

Repeat  process  until  on  adding  fresh 
alcohol,  the  fluid  only  becomes 
tinged  violet. 

Pour  ofC  alcohol,  leaving  litmus  as 
dry  as  possible,  connect  up  bottle 
to  an  air  pump  and  evaporate  off 
the  last  trace  of  alcohol. 

Transfer  the  dry  litmus  to  a  liter 
flask,  measure  in  600  cc.  distilled 
water  and  allow  to  remain  in  con- 
tact for  24  hours,  with  frequent 
shaking. 

Filter  the  solution  and  add  to  it  one 
or  two  drops  of  pure  sulphuric  acid 
until  litmus  solution  is  distinctly 
wine-red  in  color. 

Add  excess  of  pure  solid  baryta  and 
allow  to  stand  until  the  reaction  is 
again  alkaline. 

Filter. 

Bubble  CO2  through  the  solution  until 
reaction  is  acid. 

Sterilize  at  100  C.  for  30  minutes  for 
3  days.  This  also  drives  ofC  C02, 
leaving  the  solution  neutral. 

TUBING  OF  NUTRIENT  MEDIA. 

After  filtration  the  media  is  placed  in 
flasks  or  tubes.  The  flasks  and  tubes 
must  first  have  been  washed,  dried 
and  their  mouths  firmly  closed  with 
a  cotton  plug  about  1  and  i/^  inches  in 
length  (allowing  about  V2  inch  or 
more  to  extend  beyond  the  mouth). 
The  flasks  and  tubes  are  then  placed 


58  BACTERIOLOGY. 

in  the  hot-air  oven  for  about  one 
hour  at  150°  C.  which  bakes  and 
molds  the  plugs  and  sterilizes  the  ap- 
paratus. 

The  liquefiable  media  (liquefied)  and  the 
liquid  media  may  be  tubed  by  means 
of  a  pipette  attached  to  a  funnel  by 
a  short  rubber  tube  on  which  is  fitted 
a  pinch-cock  to  regulate  the  flow  of 
media  into  the  tube.  The  tube  should 
contain  about  1  and  inches  or  10  cc. 
of  media. 

A  special  apparatus  for  tubing  media 
has  been  constructed  that  allows  the 
media  to  be  measured  into  each  tube. 

Fluid  media  containing  carbohydrates 
may  be  filled  into  Smith's  fermenta- 
tion tubes  or  into  the  Durham  gas 
tubes;  the  latter  tubes  are  ordinary 
media  tubes,  having  smaller  tubes 
inverted  inside  them.  When  first 
filled  the  small  tubes  will  float  on  the 
surface,  but  after  sterilization  all  the 
contained  air  will  be  replaced  by 
media. 

The  media  must  now  be  sterilized  in 
the  Arnold  for  3  consecutive  days. 

STERILIZATION. 

Dr;jr  Heat  produced  by  means  of  a  "hot- 
air  oven"  heated  by  a  gas  burner. 
Hot  air  at  150**  C.  will  destroy  all 
bacteria  and  their  spores  in  about 
30  minutes.  An  exposure  at  about 
180°  C.  for  a  few  minutes  only  will 
do  the  same.  This  method  of 
sterilization  can  be  used  with  glass, 
metal  or  small  bulk  fabric  only. 
Large  masses  of  fabric  are  not 
readily  sterilized  by  dry  heat  on  ac- 
count of  its  poor  penetrative  power. 

MOIST  HEAT. 

Fractional  Sterilization.  A  water  bath 
with  a  temperature  of  56°  C,  if 
maintained  for  30  minutes,  will  de- 
stroy vegetative  bacteria.  It  will, 
however,  have  no  effect  on  spores. 

It  is  used  for  sterilizing  albuminous 
fluid  media  that  would  coagulate  at 
a  higher  temperature. 

Method.  The  water  bath  is  heated  by 
a  Bunsen  flame  to  56°  C.  If  the 
bath  is  not  controlled  by  a  thermo 
regulator  it  must  be  watched  care- 
fully.   The  material  to  be  sterilized 


BACTERIOLOGY. 


59 


is  now  placed  in  the  bath  so  that 
it  will  be  at  least  2  cm.  below  the 
lev'"^  of  the  water.  The  temperature 
of  t'  i  bath  will  probably  fall  some- 
whtd',  but  will  again  in  a  few 
minutes  rise  to  56''  C.  The  material 
is  removed  and  subjected  to  the 
rapid  cooling  effect  of  running" 
water.  The  vegetative  forms  are 
killed  and  it  is  now  put  for  24 
hours  in  a  cool,  dark  place,  at  the 
end  of  which  time  some  of  the 
spores  will  have  germinated  and 
assumed  the  vegetative  form,  these 
are  killed  by  a  similar  exposure  to 
56°  C.  on  the  second,  third,  fourth, 
fifth  and  sixth  day  successively. 

The  water  li)atli  at  a  temperature  of 
60°C.  for  60  minutes  is  used  in  the 
sterilization  of  "bacterins,"  or  so- 
called  "vaccines." 

One  exposure  is  all  that  is  necessary 
for  reasons  to  be  explained  later 
(see  "preparation  of  bacterins"). 

Water  bath  at  100"*  C.  (Water  sterili- 
zation) destroys  vegetative  bacteria 
almost  instantly.  Spores  are  de- 
stroyed in  from  5  to  15  minutes.  It 
is  used  for  metal  instruments,  rub- 
ber stoppers,  rubber  and  glass  tub- 
ing, etc. 

STEAM. 

Steam  at  100*  C  will  destroy  vegeta- 
tive bacteria  in  from  15  to  20 
minutes  and  the  spores  in  from  1 
to  2  hours.  The  various  culture 
media  are  sterilized  by  this  method. 

Koch's  Steam  Chest  was  constructed 
for  this  form  of  sterilization.  It  is 
a  tall,  cylindrical  vessel  divided  by 
a  perforated  diaphragm  into  an  up- 
per steam  chamber  and  a  lower 
water  chamber.  The  chest  is  heat- 
ed by  gas-burners. 

Arnold's  Steam  Sterilizer  is  a  modifi- 
cation of  Koch's  chest.  It  is  very 
efficient  and  much  used. 

Method: 

When  live  steam  issues  steadily  from 
the  sterilizer,  the  material  to  be 
sterilized  is  placed  within  the  steam 
compartment  and  allowed  to  remain 
for  20  minutes,  if  the  media  is 
liquid,  and  for   30  minutes  if  the 

*    media  is  liquefiable  or  solid. 


60 


BACTERIOLOGY. 


This  will  kill  all  vegetative  bacteria. 
During  the  hours  of  cooling  the 
spores  will  germinate  and  can  then 
be  destroyed  by  repeating  the  pro- 
cess in  24  hours.  ♦  At  the  end  of 
another  24  hours  the  media  is  sub- 
jected  to  another  sterilization. 

The  method  is  spoke]«i  of  as  a  discon- 
tinuons  or  Intermittent  sterilization. 

Continuous  Sterilization.  An  exposure 
to  steam  at  100°  C.  for  1  to  2  hours 
is  sometimes  practiced,  but  is  not 
to  be  recommended. 

SuperlieatecL  Steam.  Chamberland's 
Autoclave  consists  of  a  metallic 
cylinder  fitted  with  a  movable  lid 
which  seals  the  cylinder  by  means 
of  bolts.  It  also  has  a  manometer, 
vent  cock  and  safety  valve.  It  per- 
mits the  heating  of  steam,  under 
pressure  to  115°  C.,  ^^nd  will  destroy 
both  vegetative  bacV^r^i^ia  and  spores 
within  15  minutes.  If  the  pressure  is 
increased  so  as  to  raise  the  temper- 
ature to  120°  C,  the  vegetative  bac- 
teria and  spores  will  be  killed  in 
10  minutes. 

Although  it  is  a  short,  effective  meth- 
od of  sterilization  and  was  formerly 
employed  to  a  great  extent  for 
media,  on  account  of  hydrolytic 
•changes  in  media  subjected  to  high 
temperatures,  which  renders  it  un- 
fit for  the  cultivation  of  the  more 
delicate  organisms,  its  use  has  been 
restricted  to  disinfecting  old  cul- 
tures, contaminated  articles,  etc. 

FIZkTEBS    FOB    STEBII^IZATION  OP 
AIB  AND  I^IQUIDS. 

Cotton  wool  is  used  in  the  laboratory 
for  sterilizing  air  or  gases.  It  is 
put  as  a  loose  plug  in  a  glass  tube 
or  a  modified  tube  (air  filter)  and 
sterilized  in  the  hot  air  oven.  If 
the  cotton  plug  is  prevented  from 
becoming  moist  (from  air  or 
liquids)  it  will  prevent  organisms 
from  entering. 

Porcelain  Filters  are  used  for  steril- 
izing liquids. 

The  liquids  are  passed  through  a 
cylindrical  vessel,  closed  at  one  end 
like  a  test  tube,  made  of  either 
porous  "biscuit"  porcelain,  hard- 
burnt  and  unglazed  (Chamberland) 
or  of  Kleselguhr,  a  fine  diatoma- 


BACTERIOLOGY.  61 

ceous  earth  (Berkfield)  and  are 
called  "candle"  or  "bougies."  In 
passing  the  liquids  through  these 
candles,  the  bacteria  are  retained  in 
the  pores  of  the  filter  which  renders 
the  liquid  germ-free. 

BACTERIAL  CULTIVATION. 

Zdentlflcatlon  of  Bacteria.  Culture 
Characteristics.  Staining*. 

Aerobic  Bacteria. 

1.  Tube  Cultures,  accomplished  by  means 

of  a  straight  or  a  looped  end  plat- 
inum wire  fastened  to  the  end  of  a 
glass  or  aluminum  rod.  The  pro- 
cedure is  as  follows: 

(a)  Sterilize  the  wire  by  heating  in 

a  flame. 

(b)  Remove   cotton   plug   from  tube 

and  hold  the  end  of  plug  that 
has  not  been  within  the  tube 
between  the  fingers. 

(c)  Touch  wire  to  the  material  to  be 

transferred. 

(d)  Stroke     or     smear     (stroke,  or 

"streak"  culture,  is  made  by 
drawing  the  wire  as  lightly  as 
possible  along  the  center  of  the 
surface  of  the  medium.  "Smear" 
culture  is  made  by  rubbing  the 
loop  all  over  the  surface  of  the 
m  e  d  i  u  m)  the  contaminated 
straight  wire  over  the  surface 
of  the  slanted  media;  or  if  the 
media  is  not  slanted,  "stab" 
culture  the  solidified  media  with 
the  wire.  This  is  also  employed 
for  the  so-called  "shake  cul- 
tures." 

(e)  Replace  the  cotton  plug. 

(f)  Sterilize   the  wire   in   the  flame. 

Label  for  identification  and  with 
the  date  of  inoculation. 

(g)  Place   all   inoculated    tubes,  ex- 

cept that  containing  gelatin,  in 
the  incubator. 

2.  Plate  Cultures.  Petri  dishes  are  used. 

These  are  two  shallow  glass  dishes, 
so  made  that  one  will  cover  the 
other.  These  cultures  are  made  in 
order  that  the  appearances  of  the 
separate  colonies  may  be  studied. 
Having  first  sterilized  the  Petri  dishes 
in  a  hot  air  oven,  the  procedure  for 
plating  the  culture  is  as  follows: 


62 


BACTERIOLOGY. 


(a)  Several  tubes  of  agar  or  gelatin 

are  melted,  then  cooled  to  a 
temperature  not  destructive  to 
the  bacteria;  i2°  C.  for  agar  and 
lower  for  the  gelatin.  A  water 
bath  with  a  constant  tempera- 
ture of  about  43"  C.  is  very  con- 
venient. 

(b)  Place  3  sterile  Petri  dishes  in  a 

row  and  number  them  1,  2  and  3. 

(c)  Looped-end  wire  is  sterilized  over 

flame. 

(d)  A    loopful    of    culture    is  then 

shaken  in  the  tube  of  melted 
media.  This  tube  is  marked 
No.  1,  or  first  dilution.  Shake 
with  an  even  circular  move- 
ment so  as  to  diffuse  the  in- 
oculum throughout  the  medium. 

(e)  Sterilize  the  loop  and  transfer  2 

loopfuls  of  No.  1  to  another 
tube  of  melted  media.  This  is 
marked  No.  2,  or  second  dilu- 
tion.   Mix  as  before. 

(f )  In  like  manner  transfer  3  loopfuls 

of  No.  2  to  another  tube  of 
melted  media  and  this  is  marked 
No.  3,  or  third  dilution.  Mix  as 
before. 

(g)  Sterilize  the  wire. 

(h)  Flame  the  plug  of  tube  No.  1, 

remove  it,  flame  the  lips  of  the 
tube,  raise  the  cover  of  Petri 
dish  No.  1  and  pour  the  inocu- 
lated liquefied  medium  into  it 
so  as  to  form  a  thin  layer  over 
the  bottom  of  the  plate. 
No.  2  and  3  are  poured  in  a  sim- 
ilar manner. 
Place   agar   dishes   in   the  incu- 
bator.    The  gelatin  dishes  are 
to  remain  at  room  temperature. 
The  dilutions  are  made  in  order  that 
the   colonies   may   be   thinned  out, 
thus  allowing  their  accurate  study 
and   sometimes   their   separate  re- 
covery, when  under  various  condi- 
tions there  may  be  more  than  one 
kind  of  bacterial  colonies  present. 
In  the  pouring  of  the  plates,  No.  1 
(1st  dilution)  rarely  gives  a  plate 
of   any   value,    therefore   it   is  re- 
placed by  a  tube  of  bouillon  or  salt 
solution;  the  plate   (No.  1)   is  not 
poured. 


BACTERIOLOGY. 


63 


When  the  main  object  of  the  dilutions 
is  to  obtain  subcultivations  from  a 
number  of  individual  bacteria,  "sur- 
face plates"  are  prepared. 

Method.  Liquefy  three  tubes  of  lique- 
fiable  media  and  pour  each  tube  into 
a  separate  Petri  dish  and  allow  to 
solidify.  When  cold  place  a  drop 
of  the  inoculum  on  the  surface  of 
the  media  close  to  one  side  of  the 
plate  and  with  a  platinum  wire, 
glass  rod  or  aluminum  wire  (bend 
about  4  cm.  of  one  end  at  a  right 
angle,  sterilized)  smear  the  drop 
over  the  surface  with  the  short  arm 
of  the  spreader  (holding  the  plate 
vertical).  Rub  the  infected  spreader 
over  the  surface  of  No.  2  plate  then 
over  No.  3  plate.  Sterilize  the 
spreader,  label  and  incubate  the 
the  plates. 

ANAEROBIC  BACTERIA. 

Anaerobic  cultures  are  made  by  grow- 
ing the  organisms,  by  means  of  cul- 
ture tubes  or  plates,  in  the  absence 
of  oxygen.  This  is  accomplished 
by: — Exclusion  of  the  air  from  the 
cu4tivation;  exhaustion  of  the  air 
from  the  vessel  containing  the  culti- 
vation by  an  air  pump;  displacement 
of  air  by  an  indifferent  gas,  e.  g. 
hydrogen;  absorption  of  oxygen  by 
means  of  pyrogallic  acid  rendered  al- 
kaline with  caustic  soda  (nitrogen 
atmosphere) ;.  a  combine  of  two  or 
more  of  the  above. 

METHODS. 

Hesse's.  A  deep  stab  in  agar  or 
gelatine  is  made  with  the  needle 
containing  the  organism,  and  the 
tube  is  then  nearly  filled  with 
melted  sterile  media,  or  a  layer  of 
sterilized  oil  is  poured  upon  the  sur- 
face 1  to  2  cm.  deep. 

Another  method,  when  dealing  with 
pure  cultivation  is  to  make  a  plate 
of  agar  or  gelatin,  inoculate  one 
spot  of  the  surface  and  place  over 
the  spot  a  sterile  cover  slip  or 
piece  of  mica,  well  pressed  down  to 
exclude  air  bubbles. 

Roux's.  Aspirate  inoculated  media 
into  capillary  pipettes  and  seal  each 
end  of  pipette  in  a  blow  flame. 


BACTERIOLOGY. 


Another  method,  sometimes  spoken  of 
as  the  "biological  method,"  is  to 
make  a  deep  stab  into  gelatin  or 
agar  and  then  pour  a  layer  of  a 
broth  cultivation  of  a  vigorous 
aerobe  over  it. 

Buchner's.  An  inoculated  culture  tube 
is  placed  within  a  larger  tube,  the 
lower  part  of  which  contains  an 
alkaline  solution  of  pyrogallic  acid. 
The  tube  is  closed  with  a  rubber 
stopper.  Use  1  gm.  of  pyrogallic 
acid  for  every  100  cc.  of  air  capac- 
ity of  the  larger  tube. 

Wrigrht's.     Make  a   tube  cultivation, 

•  cut  off  the  projecting  part  of  cot- 
ton plug,  push  the  plug  into  the 
tube  (2  to  3  cm.  distance);  with  a 
pipette  run  about  1  cc.  of  a  10% 
solution  of  pyrogallic  acid  onto  the 
plug;  with  another  pipette  an  equal 
amount  of  soda  and  close  the  tube 
quickly  with  a  rubber  stopper. 

Exhaustion  of  Air.  Make  a  tube  cul- 
tivation, replace  the  cotton  plug 
with  a  perforated  rubber  stopper,  fit 
in  a  glass  tube  bent  at  a  right 
angle  with  a  construction  of  about 
3  cm.  above  the  stopper,  connect 
glass  tube  with  a  water  or  air 
pump  (interposing  a  wash-bottle 
containing  sulphuric  acid),  exhaust 
the  air  and  seal  the  glass  tube  at 
the  construction,  using  a  blow-pipe 
flame,  before  disconnecting  the 
pump. 

Kovy's.  Place  cultivations  inside 
Novy's  jar,  connect  up  delivery  tube 
with  hydrogen  apparatus,  attach 
rubber  tubing  to  exit  tube,  collect 
sample  of  issuing  gas  (over  water) 
for  testing;  when  air  is  completely 
displaced  turn  the  stopper  to  close 
entry  and  exit  tubes  and  disconnect 
the  gas  apparatus. 

Bnlloch's.  Place  cultivations  in  a 
glass  dish  resting  in  the  center  of  a 
glass  slab;  put  pyrogallic  acid  at 
one  side  of  the  dish;  put  sodium 
hydroxide  near  the  pyrogallic  acid; 
smear  flange  of  Bulloch's  jar  with 
resin  ointment;  smear  stop-cocks 
with  resin  ointment;  connect  short 
tube  with  gas  supply:  open  both 
stop-cocks;  connect  a  piece  of  glass 
tubing  by  means  of  a  piece  of  rub- 


BACTERIOLOGY. 


65 


ber  tubing  (with  a  screw-clamp)  to 
the  long  tube;  collect  issuing  gas 
and  test;  when  air  is  displaced  shut 
off  stop-cock  of  entry;  shut  off  stop 
cock  of  exit,  screw  down  clamp;  re- 
move glass  tube  from  rubber  con- 
nection; connect  up  short  tube  to 
air  pump;  open  stop-cock  of  short 
tube;  aspirate  small  quantity  of 
gas;  shut  off  stop  cock;  disconnect 
air  pump;  fill  10  cc.  bulb  pipette 
with  water  and  insert  it  into  rub- 
ber tubing  on  long  tube  as  far  as 
screw  clamp;  open  screw  clamp; 
run  in  water  till  stopped  by  in- 
ternal pressure  and  shut  off  stop 
cock.  Incubate. 
Botkins.  Place  a  leader  cross  in  a 
glass  dish  (20  cm.  in  diameter,  8 
cm.  deep)  put  tube  cultivation  in  a 
glass  jar  or  plate  cultivations  in  a 
wire  frame  resting  them  on  the 
cross,  adjust  U-shaped  pieces  of 
glass  tubing  in  a  vertical  position 
on  opposite  sides  of  the  dish,  place 
a  bell  jar  over  the  cultures  enclos- 
ing one  arm  of  each  U-tube  (resting 
it  on  the  cross),  fill  the  dish  with 
glycerine  or  mercury  to  a  depth  of 
about  5  cm.  and  connect  one  U-tube 
with  a  gas  apparatus. 

IDENTIFICATION  OF  BACTERIA. 

In  order  to  identify  an  organism  after 
isolation  it  must  be  studied  as  to 
cultural  characters,  morphology,  chem- 
ical products  of  growth,  biology  and 
pathogenicity,  as  no  microorganism 
can  be  identified  by  any  one  character 
or  property. 

STUDY  OP  GROWTH  CHARACTER- 
ISTICS.    (MICROSCOPIC  METHOD). 

Plate  Cultures.  In  gelatin  note  the 
presence  or  absence  of  liquefaction 
in  the  surrounding  medium.  Note 
the  shape  and  character  of  the 
liquefaction,  if  present. 

In  the  agar,  no  liquefaction  takes 
place.  The  liquid  found  on  the  sur- 
face is  merely  the  water  of  con- 
densation. 

The  colonies  are  to  be  examined  at 
intervals  of  24  hours, — with  the 
naked  eye,  with  a  hand  lens,  and 
under  low  power  microscope  or  dis- 


BACTERIOLOGY. 


secting.  microscope.  Distinguish  the 
superficial  from  the  deep  colonies 
and  note  the  characters  of  the 
.colonies,  as  to: — 

1.  Size.    Diameter  at  various  ages. 

2.  Shape.   Functifonu  (minute,  hemi- 

spherical) ;  round;  elliptical  (oval)  ; 
irreg-ular  (no  recognized  shape); 
fusiform  (spindle-shaped);  coch- 
leate  (like  snail  shell);  amoeboid 
(streaming,  irregular);  mycelioid 
(mold-like);  filamentous  (irreg- 
ular mass  of  filaments) ;  floccose 
(dense,  wooly  structure);  rhizoid 
(root-like) ;  conglomerate  (aggre- 
gate of  colonies  of  similar  size 
and  form);  toruloid  (aggregate  of 
of  colonies  like  budding  of  yeast) ; 
and  rosulate  (rosette-like). 

3.  Surface  Elevation. 

(a)  Creneral  character  of  surface  as 

a  whole.  Flat  (thin,  leafy, 
spread  over  the  surface) ;  ef- 
fused (spread  over  the  sur- 
face as  a  thin,  veilly  layer, 
more  delicate  than  the  "flat") ; 
raised  (growth  thick,  with 
abrupt,  terraced  edges);  con- 
vex (surface  the  segment  of 
a  circle  but  very  flatly  con- 
vex); pulvinate  (surface  the 
segment  of  a  circle,  but  de- 
decidedly  •  convex);  capitate 
(surface  hemispherical);  um- 
bilicate,  (having  a  central  pit 
or  depression);  conical  (cone 
with  rounded  apex) ;  and  um- 
bonate  (having  a  central  con- 
vex, nipple-like  elevation). 

(b)  Detailed  characters  of  Surface. 

Smooth  (surface  even);  al- 
veolate (marked  by  depres- 
sion separated  by  thin  walls 
so  as  to  resemble  a  honey- 
comb); punctate  (dotted  with 
punctures  like  pin-pricks) ; 
bullate  (like  a  blistered  sur- 
face rising  in  convex  prom- 
inences, rather  coarse) ;  vesic- 
ular (more  or  less  covered 
with  minute  vesicles  due  to 
gas  formation,  more  minute 
than  the  "bullate");  verrucose 
(wart-like,  bearing  wart-like 
prominences) ;  squamose 
(scaly) ;  echinate  (beset  with 


BACTERIOLOGY. 


67 


pointed  prominences) ;  papil- 
late (beset  with  nipple  or 
mamma-like  processes);  ru- 
gose (short,  irregular  folds, 
due  to  shrinkage  of  surface 
growth);  corrugated  (in  long 
folds,  due  to  shrinkage) ;  con- 
toured (an  irregular  but 
smoothly  undulating  surface, 
resembling  the  surface  of  a 
relief  map);  and  rimose 
(abounding  in  chinks,  clefts 
cracks). 

4.  Internal  Structure  of  Colony.  (Mi- 
croscopic). 

Refraction  weak:  Outline  and  sur- 
face of  relief  not  strongly  defined. 

Refraction  strong:  Outline  and 
surface  of  relief  strongly  defined; 
dense,  not  filamentous  colonies. 

(a)  General.    Amorphous  (without 

any  definite  structure) ;  hy- 
aline (clear  and  colorless); 
homogeneous  (structure  uni- 
form) ;  and  homochromous 
(color  uniform). 

(b)  Granulations     or  Blotching's. 

Finely  granular;  coarsely 
granular;  grumose  (coarser 
than  the  preceding,  with  a 
clotted  appearance,  and  part- 
icles in  clustered  grains) ; 
moruloid  (having  the  char- 
acter of  a  mulberry,  segment- 
ed, by  which  the  colony  is  di- 
vided in  more  or  less  regular 
segments);  and  clouded  (hav-. 
ing  a  pale  ground,  with  ill- 
defined  patches  of  a  deeper 
tint). 

(c)  Colony    Marking"    or  Stripingf. 

Reticulate  (in  the  form  of  a 
network,  like  the  veins  of  a 
leaf);  areolate  (divided  into 
rather  irregular,  or  angular 
spaces  by  more  or  less  definite 
boundaries);  gyrose  (marked 
by  wavy  lines,  indefinitely 
placed) ;  marmorated  (show- 
ing faint,  irregular  stripes,  or 
traversed  by  vein-like  mark- 
ings, as  in  marble) ;  rivulose, 
(marked  by  lines  like  the 
rivers  of  a  map)  and  rimose 
(showing  chinks,  cracks,  or 
clefts). 


68 


BACTERIOLOGY. 


(d)  Pilamentous  Colonies.  Fila- 
mentous; floccose  (composed 
of  filaments,  densely  placed); 
and  curled  (filaments  in 
parallel  strands,  like  locks  or 
ringlets). 

5.  Hdges  of  Colonies.    Entire  (without 

toothing  or  division);  undulate 
(wavy);  repand  (like  the  border 
of  an  open  umbrella) ;  erose  (as  if 
gnawed);  irregular  (toothed);  lob- 
ate;  lobulate  (minutely  lobate); 
auriculate  (with  ear-like  lobes) ; 
lacerate  (irregularly  cleft,  as  if 
torn) ;  fimbriate  (fringed)  ;  ciliate 
(hair-like  extensions,  radiately 
placed);  tufted;  filamentous  and 
curled. 

6.  Optical  Characters  (after  Shuttle- 

worth). 

(a)  General     Characters.  Trans- 

parent (transmitting  light); 
vitreous  (transparent  and  col- 
orless); oleaginous  (trans- 
parent and  yellow,  olive  to 
linseed-oil  colored) ;  resinous 
transparent  and  brown,  varn- 
ish or  resin-colored) ;  trans- 
lucent (faintly  transparent) ; 
porcellaneous  (translucent  and 
white);  opalescent  (trans- 
lucent, greyish-white  by  re- 
flected light) ;  nacreous  (trans- 
lucent, greyish-white,  with 
pearly  lustre);  sebaceous 
(translucent,'  yellowish  or 
greyish-white);  butyrous 
(translucent  and  yellow); 
ceraceous  (opaque  and  white, 
chalky);  dull  (without  lust- 
er); glistening  (shining); 
fluorescent  and  iridescent. 

(b)  Chromog'enicity.    Color  of  pig- 

ment,   pigment   restricted  to 
colonies,  pigment  resticted  to 
medium  surrounding  colonies 
and  pigment  present  in  col- 
onies and  in  medium. 
Streak  or  Smear  Cultures.    In  gelatin 
and  agar  note  the  points  as  indi- 
cated   under    plate   cultures.  In 
blood  serum  note  the  presence  or 
absence  of  liquefaction. 
Gelatin  Stab  Cultures.    Note  as  to 
1.  Surface  Growth.    Same  as  in  plate 
cultures. 


BACTERIOLOGY. 


69 


2.  l^ine  of  Puncture.     Filiform  (uni- 

form growth) ;  nodose  (closely 
ag-g^regated  colonies);  beaded 
(loosely  placed  or  disjointed 
colonies);  papillate  (beset  with 
papillate  extensions);  echinate 
(beset  with  acicular  extensions); 
villous  (beset  with  short,  undi- 
vided, hair-like  extensions); 
pulmose  (a  delicate,  feathery 
growth) ;  arborescent  .(branched 
or  tree-like,  beset  with  branched 
hair-like  extensions). 

3.  Area  of  Iiiquef action  (if  present). 

Crateriform  (a  saucer-shaped 
liquefaction);  saccate  (shape  of 
an  elongated  sack,  tubular,  cyl- 
indrical); infundibuliform  (shape 
of  a  funnel,  conical);  napiform 
(shape  of  a  turnip);  fusiform 
(outline  of  a  parsnip,  narrow  at 
either  end,  broadest  below  the 
surface) ;  and  stratiform  (lique- 
faction extending  to  the  walls  of 
the  tube  and  downward  horizon- 
tally). 

4.  Character  of  tlie  I^iquefied  Gelatin. 

Pellicle  on  surface;  uniformly 
turbid;  granular;  mainly  clear, 
but  containing  flocculi;  deposit  at 
apex  of  liquefied  portion. 

5.  Production  of  Gas  Bubbles. 
SHAKE  CUXiTUBBS.  Presence  or  ab- 
sence of  liquefaction;  production  of 
gas  bubbles;  bulk  of  growth  at  the 
surface  (aerobic) ;  bulk  of  growth  in 
depths  (anaerobic). 

FI^UID  MEDIA. 

1.  Surface  of  the  Liquid.  Presence 

or  absence  of  froth  due  to  gas 
bubbles;  presence  or  absence  of 
pellicle  formation;  character  of 
pellicle. 

2.  Body   of   the   LiCLuid.  Uniformly 

turbid;  flocculi  in  suspension; 
granules  in  suspension;  clear, 
with  precipitate  at  the  bottom  of 
tube;  coloration  of  fluid,  presence 
or  absence. 

3.  Precipitate.      Character;  amount; 

color. 

CARBOHYDRATE  MEDIA.  Note: 
Growth;  reaction;  gas  formation. 

lilTMUS  MII^K  CULTIVATIONS.  Note: 
Reaction  (unaltered,  acid  or  alkaline) ; 
odor;  gas  formation;  consistency  (un- 


70 


BACTERIOLOGY. 


altered,  peptonized  or  coagulated) ; 
clot  (solid,  flocculent  or  rag-ged  and 
broken  up  by  gas  bubbles;  coagulum 
undissolved;  coagulum  finally  pepton- 
ized, completely  or  incompletely;  re- 
sulting solution,  clear  or  turbid) ; 
whey  (abundant  or  scanty,  clear  or 
turbid,  coagulated  by  boiling  or  not). 

STUDY  .OF  BACTERIA  BY  MICRO- 
SCOPIC METHODS. 
I.IVING  BACTERIA:    Note  motility  or 
non-motility.    If  the  organism  is  one 
which    forms    spores  observe — spore 
formation  and  spore  germination. 
METHODS  OF  EXAMINATION. 
1.  Ordinary  Method. 

If  specimen  from  solid  media  is 
used,  a  drop  of  water  is  placed 
on  a  clean  slide. 
If  specimen  from  liquid  media  is 
used,  a  drop  of  the  media  con- 
taining the  bacteria  is  used. 

(a)  Flame  the  cotton  plug  of 
tube  containing  the  culture;  ex- 
tinguish the  burning  cotton. 

(b)  Hold  test  tube  containing  cul- 
ture between  thumb  and  finger  of 
left  hand. 

(c)  Hold  platinum  needle  between 
thumb  and  forefinger  of  right 
hand,  and  sterilize  by  heating  red 
hot.    Allow  to  cool. 

(d)  Remove  cotton  plug  with  the 
third  and  fourth  finger;  insert 
needle,  and  transfer  minute  por- 
tion of  the  bacterial  culture  to  the 
slide. 

(e)  Return  plug  to  tube  and  ster- 
ilize needle. 

(f)  Place  a  clean  cover  glass  over 
specimens  and  examine  first  with 
1-6  objective,  then  with  1-12  of 
oil  immersion  pushed  gently  into 
a  drop  of  cedar  oil  placed  on  the 
cover  glass.  Use  the  fine  adjust- 
ment. Examination  should  be 
made  by  dim  light. 

During  the  examination,  stains  and 
other  regents  may  be  run  in 
under  the  coverslip.  The  non- 
toxic basic  dyes  for  **intra-vitam" 
staining  of  bacteria  are  neutfal, 
red,  quinoline  blue,  methylene 
green  and  vesuvian  in  0.5% 
aqueous  solutions. 


BACTERIOLOGY. 


71 


2.  Burris'    Negrative    Stain    is  some- 

times employed  to  simulate  dark 
g^round  illumination.  It  is  pre- 
pared by  mixing  25  cc.  of  liquid 
black  ink  (any  liquid  waterproof 
black  drawing  inks)  and  1  cc.  of 
tincture  of  iodine.  Allow  the  mix- 
ture to  stand  for  24  hours,  cen- 
trifugalize,  pipette  off  the  super- 
natant liquid  to  a  clean  bottle, 
and  then  a  crystal  of  thymol  or 
1  drop  of  formalin  as  a  preserva- 
tive. 

'With  a  sterilized  loop  place  one 
drop  of  the  liquid  ink  close  to  one 
end  of  a  slide;  sterilize  the  loop 
and  place  a  drop  of  the  fluid  cul- 
ture (or  emulsion  of  solid  cul- 
ture) on  the  slide  by  the  side  of 
the  ink;  mix  thoroughly;  sterilize 
the  loop;  with  another  slide 
spread  the  mixture  across  the 
slide  (by  placing  the  end  of  the 
slide  used  as  a  spreader  trans- 
versely and  at  an  angle  of  about 
60°  on  the  mixture  and  allow  the 
fluid  to  spread  across  the  slide 
and  fill  the  angle  of  incidence; 
draw  it  toward  the  end) ;  dry  in 
the  air  and  examine  with  1/12  oil 
objective. 

3.  "Hanging"  drop"  method. 

(a)  Paint  a  ring  of  vaseline  around 
the  hollow  in  a  "culture  slide." 

(b)  Place  bacterial  culture  in  a 
small  drop  on  a  clean  cover  glass. 

(c)  Invert  slide  over  the  cover 
glass,  the  drop  to  be  within  the 
vaseline  ring,  but  not  to  touch  its 
sides,  and  press  down  so  as  to 
seal  tight. 

(d)  Invert  carefully  and  examine. 
This  method  is  for  demonstration 
of  bacterial  motility.  It  may  be 
kept  for  examination  from  day  to 
day  s6  that  spore  formation  and 
germination  can  also  be  studied. 

To  Study  Spore  F6miation.  Prepare  the 
hanging  drop  from  vegetative  forms, 
add  a  trace  of  0.5%  magenta  solution 
to  render  bacilli  more  distinct,  place 
slide  under  microscope  (using  a  warm 
stage  if  necessary) ;  with  the  1/6  lens 
select  a  bacillus  for  observation,  then 
substitute  the  1/12  oil  immersion  and 
observe  the  formation  of  the  spore. 


72 


BACTERIOLOGY. 


To  Study  Spore  Germination.  Prepare 
the  hanging  drop  from  old  cultiva- 
tions in  which  no  living  vegetative 
forms  are  present  and  observe  the 
process  of  germination  as  in  "spore 
formation." 

FIXED  AND  STAINED  BACTERIA. 
Bacteria  are  rendered  more  prominent 
by  the  use  of  dyes  and  by  their  aid, 
note — 

1.  The  points   in   morphologfy,   as: — 

Shape,  size  and  pleomorphism 
if  present,  record — predominant 
character  of  the  variant  forms, 
the  media  on  which  they  are  ob- 
served, at  what  period  of  develop- 
ment). 

2.  The    details    of    structure,    as: — 

Flagella  (if  present,  record — 
method  of  staining,  position,  ar- 
rangement and  number) ;  spores 
(if  present,  record — method  of 
staining,  shape,  size,  position 
within  cell,  condition  as  to  shape 
of  parent  cell,  optimum  medium 
and  temperature,  age  of  cultiva- 
tion, condition  of  environment  as 
to  temperature  and  atmosphere, 
methods  of  germinations);  invo- 
lution forms  (if  present,  record — 
method  of  staining,  character 
e.  g.  living  or  dead),  shape,  on 
what  medium  observed,  age  of 
medium  and  environment) ;  me- 
tachromatic granules  (if  present, 
record — method  of  staining,  char- 
acter of  granules,  number  of 
granules  and  color  of  granules). 
REACTION  OF  STAINS. 

1.  Gram's  Method.    Positive  (not  de- 

colorized) or  negative  (decolor- 
ized.) 

2.  Neisser's  Method.    If  granules  are 

present,  record  their  position  and 
number. 

3.  Ziehl-Neelsen's  Method.  Acid-fast 

or  decolorized. 

4.  Simple  Aniline  Dyes.    Record  those 

giving  best  results. 

STRAINING  METHODS. 

Most  bacteria  stain  easily  and  are  there- 
fore easily  decolorized. 

Some  bacteria  can  withstand  alcohol 
and  some  withstand  strong  solutions 
of  mineral  acids  without  decolorizing. 


BACTERIOLOGY. 


73 


It  is  often  necessary  to  use  heat  or  a 
mordant  in  order  that  the  stain  may 
penetrate  the  cell. 

Potassium  hydrate,  aniline  oil,  alcohol, 
carbolic  acid  (1-5%)  and  acetic  acid 
(1-5%)  are  the  common  mordants 
used. 

Foxrmtaas  of  Staining'  Solution. 

1.  Simple  aniline  stains  are  prepared 

by  saturating"  alcohol  with  methy- 
lene blue,  dahlia,  fuchsin,  Vesu- 
vian,   gentian  violet  or  thionine. 

To  these  stock  solutions  alcohol  may 
be  added  from  time  to  time,  al- 
ways allowing  an  excess  of  un- 
dissolved stain  to  remain  on  the 
bottom  of  the  vessel. 

When  required  for  use  add  5  cc.  of 
the  saturated  alcohol  solution  to 
95  cc.  of  distilled  water  and  filter. 

The  methylene  blue  stain  is  the 
only  one  that  is  permanent.  The 
others  must  be  made  fresh  as 
required  for  use. 

All  stains  should  be  filtered  before 
using,  unless  otherwise  specified. 

2.  Aniline-Gentian  Violet. 

Anilene  Water  10  parts. 

(Aniline  oil  5  cc.  and  distilled 
water  100  cc.  are  well  shaken  to- 
gether and  filtered.  Make  fresh 
every  time). 
Saturated  alcoholic  solution  of  g-en- 
tian  violet   1  part. 

3.  Aniline-fuchsin. 

Aniline  water  (see  Aniline  Gentian 
V.)    10  parts 

Saturated  alcoholic  solution  of 
fuchsin   1  part. 

4.  Alkaline  Methylene  Blue. 

(a)  IiOe£B.er's. 

Sat.    ale.    sol.     methylene  blue 

 30  parts. 

Sol.  potass,  hydrate  (1-10,000) 
  100  parts. 

(b)  Kochs. 

Sol.  potass,  hydrate  (10 

per  cent)   0.2  part. 

Sat.   ale.    sol.  methyl. 

blue    1.0  part. 

Water   (distilled)  200.0  parts. 

5.  Carbolic  acid  solutions. 

(a)  Kuhne's  Methylene-blue. 

Methylene  blue   1.5  gm. 

Abs.  alcohol    10.0  cc. 


74  BACTERIOLOGY. 

Carbolic    acid  solution 

(1-20)   100  parts. 

Stain  for  5  minutes, 
(b)  Ziehl's  carbol  fuclLsln. 

Basic  fuchsin   1  part. 

Abs.  alcohol  10  parts. 

Carbolic     acid  solution 

(1-20)   100  parts. 

Filter. 

6.  Gram's  Iodine  Solutioxi. 

Iodine    1  part. 

Potass,  iodid    2  parts. 

Distilled  water   300  parts. 

7.  Gab'bet's  Acid  Blue  (a  rapid  stain). 

25  per  cent  solution  of 

sulphuric  acid   100  parts. 

Methylene  blue    2  parts. 

Allow  dilute  acid  to  stand  24  hours 
before  adding  the  methylene  blue. 

8.  Unna's  Borax  Methyl  Blue. 

Borax   1  part. 

Methyl  blue    1  part. 

Water   100  parts. 

9.  Unna's  Polyclirome  Methylene  Blue. 

Potassium  carbonate  ...     1  piart. 

Methylene  blue    1  part. 

Water   100  parts. 

Must  be  ripened  for  months. 

10.  NicoUe's  Carhol-thionine. 

Sat.  sol.  thionine  in  ale.  (90 

per  cent)    10  cc. 

Aqueous  sol.  ac.  carbol.  (1 

per  cent)   100  cc. 

Stain  sections  one-half  to  one  min- 
ute. 

Contrast  Stains. 

11.  Eosin  Aqueous  Solution. 

Bosin  (aqueous)    1  gm. 

Water  (distilled)   100  cc. 

Absolute  alcohol    5  cc. 

12.  Bosin  Alcoholic  Solution. 

Eosin    (alcoholic)   0.5  gm. 

Alcohol  (70%)   100  cc. 

13.  Safranine  Aqueous  Solution. 

Safranine   0.5  g. 

Water  (distilled)   100  cc. 

14.  Neutral  Bed  Aqueous  Solution. 

Neutral  red   1.0  gm. 

Water  (distilled)   100  cc. 

15.  Vesuvin  (or  Bismarck  Brown). 

Vesuvin   0.5  gm. 

Water  (distilled)  100  cc. 

Special  Stains. 

16.  MacConkey's  Stain  (for  capsules). 

Dahlia   0.5  gm. 


BACTERIOLOGY. 


75 


Methyl  green  (crystals)  .  .  1.5  gm. 

Water  (distilled)   100  cc. 

Mix  well  in  mortar,  then  add 

Fuchsin  (sat.  alcohol  sol.).  10  cc. 
Water  (distilled)  to  make..  200  cc. 

17.  Muir's  Mordant  (for  capsules). 

Mercuric  bichloride   (sat.  aq. 

sol.)   2  cc. 

Tannic  acid  (20%  aq.  sol.).  .  .2  cc. 
Potash  alum.  (sat.  aq.  sol.)..5cc. 

18.  Biblsert's  Stain  (for  capsules). 

Axjetic  acid  (glacial)   12.5  cc. 

Alcohol  (absolute)    50.0  cc. 

Water  (distilled)   100.0  cc. 

Warm  to  36°  C.  and  saturate  with 
dahlia. 

19.  Muir's  Mordant  (for  flaffella). 

Tannic  acid  (10%  aq.  sol.)..10cc. 

Mercuric  bichloride  (sat.  aq. 

sol.)    5  cc. 

Alum.  (sat.  aq.  sol.)   5  cc. 

Carbol  fuchsin  (Ziehl)   5  cc. 

Allow   to   settle   for  a  few  hours, 

decant    off    the    clean    fluid  into 

tubes  and  centrifugalize. 
It  will  keep  for  a  couple  of  weeks, 

but  is  at  its  best  4  or  5  days  after 

its   manufacture.     Must   be  cen- 

trifugalized  each  time  before  use. 

20.  l^oeffler's  Mordant  (for  flagrella). 
Tannin  (20%  aq.  sol.)  ....  10  parts. 
Ferrous  Sulphate  (sat.  aq. 

sol.)    5  parts. 

Decoc.  of  logwood  (1  to  8 

aq.  sol.)    3  parts. 

Carbolic  acid  (1%  aq.  sol.)  4  parts. 
Must  be  freshly  prepared. 

21.  IiOe£B.er's  Stain  (for  flagrella). 

Methylene-blue   4  gms. 

Aniline  water  (freshly  satu- 
rated and  filtered)  100  cc. 

or — 

Methyl ene-violet   4  gms. 

Freshly  saturated  and  filtered 
Aniline  water  (freshly  satu- 
rated and  filtered)  100  cc. 

or — 

Fuchsin   4  gms. 

Aniline  water  (freshly  satu- 
rated and  filtered)  100  cc. 

22.  Fitfield's   Mordant    (for  flagrella). 

Tannic  acid  1  gm. 

Water   10  cc. 

23.  Pitfield  Stain, 

Alum.  (sat.  aq.  sol.)  10  cc. 


76 


BACTERIOLOGY. 


Gentian  violet  (sat.  ale.  sol.)  .  1  cc. 
Water   (distilled)   5  ce. 

24.  Van  Ermengrem's  Fixing-  Plnid  (for 
flagella). 

Osmic  acid  (2%  aq.  sol.)  .  . .  .  10  cc. 
Tannic  acid  (20%  aq.  sol.) .  .  .20  cc. 

Acetic  acid  (glacial)   1  cc. 

Prepare  a  few  days  before  using-. 
Filter  when  needed.  It  should  be 
violet  in  color. 

25.  Van  Ermengrem's  sensitising"  Solu- 
tion (for  flagella). 

Silver  nitrate  (0.5%  aq.  sol?) 
Keep  in  the  dark  and  filter  just 
before  using. 

26.  Van  Ermengem's  Reducing  solu- 
tion (for  flagella). 

Gallic  acid   5  gms. 

Tannic  acid   3  gms. 

Potassium  acetate  (fused)*10  gms. 

Water  (distilled)  ...350  cc. 

Prepare  fresh  and  filter. 

27.  Bung-e's  Mordant  (for  flagella). 
Tannic  acid  (20%  aq.  sol.)..10cc. 
Ferrous    sulphate    (sat.  aq. 

sol.)    5  cc. 

Fuchsin  (sat.  ale.  sol.)   1  cc. 

28.  Pappenheini's  Corallin,  Methylene- 
.    blue  Solution  (for  B.  tuberculosis). 

Corallin   1  gm. 

Methylene-blue    (sat.  alco. 

sol.)   100  cc. 

Glycerine   30  cc. 

29.  Spengler's  picric  acid  alcohol. 

Alcohol  (absolute)   20  cc. 

Picric  acid  (sat.  aq.  sol.)  .  .  .  .10  cc. 
Water  (distilled)   10  cc. 

30.  Neisser's  Stain  (for  diphtheria) 

SOLUTION  I. 

Methylene-blue    1  gm. 

Alcohol  (96%)    20  cc. 

Dissolve  and  add 

Water  (distilled)   950  cc. 

Acetic  acid  (glacial)   50  cc. 

SOLUTION  II. 

Vesuvian    2  gms. 

Water    (distilled)   1000  cc. 

31.  Modified  Niesser's  Stain  (for  diph- 
theria). 

SOLUTION  L 
Methylene-blue    (sat.  ale. 

sol.)     4  cc. 

Acetic  acid  (5%  aq.  sol.)..     96  cc. 

SOLUTION  II. 

Neutral  red   2.5  gms. 

Water  (distilled)    1000  cc. 


BACTERIOLOGY. 


77 


m.{ 


32.  Elirlich's     Haematoxylin  (tissue 
staining). 

J   (  Haematoxylin   2  gms. 

^'     Alcohol  (absolute)   100  cc. 

Ammonium  alum  2  gms. 

Water  (distilled)   100  cc. 

Mix  I.  and  II.  stand  for  48  hours, 
then  filter  and  add 

Glycerine   85  cc. 

Acetic  acid  (glacial)  10  cc. 

Expose  to  the  light  for  ong  month 
then  filter. 

33.  Mayer's  Haematin    (tissue  stain- 
ing) 

-,-  /  Haematin    1  gm. 

^-  \  Alcohol  (909c  warmed  to 

(37°  C.)    50  cc. 

11.^  Potash  alum.  50  gms. 

I  Water   (distilled)    100  cc. 

Pour  the  two  solutions  slowly  and 

simultaneously    into    a   flask  by 

means  of  a  large  funnel  to  insure 

thorough  mixing. 

34.  Mayer's    Alum    Carmine  (tissue 

staining). 

Alum   2.5  gms. 

Carmine    1  gm. 

Place  in  a  beaker  on  a  sand  bath 
and  add  successive  small  quanti- 
ties of  distilled  water;  keep  mix- 
ture boiling  for  20  minutes.  The 
solution  should  make  up  to  100  cc. 
Filter. 

35.  Picrocarmine.    (tissue  staining). 

Picrocarmine  2  gms. 

Water  (distilled)   100  cc. 

TECHNIQUE  FOR  ORDINABY 
STRAINING. 

(a)  Prepare  clean  cover  glass  and 
slide. 

(b)  Place  drop  of  water  on  glass  or 
slide. 

(c)  Transfer  with  sterile  needle  a 
minute  portion  of  culture  to  the 
drop  of  water  and  spread  evenly 
over  surface  of  glass. 

(d)  Allow  film  to  dry. 

(e)  Fix  by  passing  the  glass  3  times 
through  a  Bunsen  flame. 

(f)  Cover  specimen  with  a  stain.  Al- 
low it  to  stain  from  2  to  10 
minutes. 

(g)  Wash  in  water. 

(h)  Dry  and  mount  in  balsam, 

(i)  Examine  with  1-12  oil  immersion 


78 


BACTERIOLOGY. 


— use  Abbe  condenser.     If  speci- 
men is  good,  label  and  preserve. 
TECHNIQUX:     FOB  DIFFEBEXTTIAI^ 
STAINING. 

1.  Gram's  Method.  (Gram's  stain). 
This  is  a  differential  stain  the  value 
depending  upon  the  mycoprotein  of 
\  certain  bacteria  forming  with  ^ini- 
line  dyes  and  an  iodid,  a  compound 
insoluble  in  alcohol.  Such  organ- 
isms are  said  to  **stain  by  Gram" 
or  to  be  "Gram  positive." 

(a)  Stain  specimen  for  5  minutes  in 
aniline  gentian-violet. 

(b)  Wash  in  water. 

(c)  Stain  with  Gram's  iodine  solution 
for  1  minute,  or  until  the  film  is 
black  or  dark  brown. 

(d)  Wash  in  95  per  cent  alcohol  until 
no  more  color  comes  away. 

(e)  Dry.  and  contract-stain  in  Bis- 
marck-brown (2-3  minutes)  or 
eosin  (1  minute). 

This  step  may  be  omitted  when  or- 
ganisms are  in  pure  culture. 

(f)  Wash,  dry  and  mount. 

The  important  bacteria  retaining  the 

stain  are  (gram  positive). 

Smegna  bacillus 

Anthrax  bacillus 

Tubercle  bacillus 

Tentani  bacillus 

Leprae  bacillus 

Diphtheria  bacillus 

Rhinoscleromatis  bacillus 

B.  Aerogenes  capsulatus 

B.  Botulinus 

B.  Subtilis 

Staphylococcus 

Streptococcus 

Pneumococcus 

Micrococcus  tetragenus 

Urethra-coccus. 
The    important    bacteria    that  de- 
colorize are  (gram  negative). 

Gonococcus 

Displococcus  intracellularis 

B.  Mucosus  capsulatus 

Bacillus  typhoid 

Bacillus  coli 

B.  Enteritidis 

Bacillus  mallei 

Bacillus  influenza 

B.  Proteus 

B.  Morax-Axenfeld 

B.  Malignant  oedema 


BACTERIOLOGY. 


79 


B.  Pyocyaneus 
Bubonic  plague  bacillus 
Koch-Weeks's  bacillus 
Cholera-asiatica  spirillum 
Micrococcus  catarrhalis 
Paratyphoid  bacillus 
Dysenteric  bacillus 
Fecal  alkalligenes  bacillus 

2.  Gram-Claudius  Method. 

(a)  Stain  in  methyl  violet  (1%  aq. 
sol.)  for  3  to  5  minutes. 

(b)  Treat  twice  with  picric  acid  (sat. 
aq.  sol.) 

(c)  Wash  in  water  and  dry. 

(d)  Decolorize  with  clove  oil. 

(e)  Wash  in  xylol. 

(f)  Mount  in  xylol  balsam. 

3.  Gram-Weig'ert  Method. 

(a)  Stain  for  5  minutes  with  aniline 
gentian  violet. 

(b)  Wash  in  water. 

(c)  Stain  with  Gram's  iodine  solution 
for  1  minute  or  until  the  film  is 
black  or  dark  brown. 

(d)  Wash  in  water  and  dry  in  air. 

(e)  Wash  in  aniline  oil  (1  part)  and 
xylol  (2  parts)  until  no  more 
color  come  away. 

(f)  Wash  in  xylol. 

(g)  Mount  in  xylol  balsam. 

4.  Ziehl-Neelsou  Method  (for  B  Tuber- 

culosis and  other  acid-fast  bacilli). 

(a)  Prepare  films  as  usual. 

(b)  Stain  in  carbol-fuchsin,  steaming, 
but  not  boiling,  for  5  minutes; 
cool  for  25  minutes. 

(c)  Wash  in  25%  sulphuric  acid  for 
3  to  5  seconds. 

(d)  Wash  in  water  (Faint  red  color 
returns). 

(e)  Wash  in  alcohol  till  no  more 
color  comes  away. 

(f)  Wash  in  water. 

(g)  Counterstain  in  weak  methylene 
blue. 

(h)  Wash  in  water,  dry,  and  mount. 
The  lepra  bacillus  and  the  smegma 

bacillus  also  stain  by  this  meth- 
od. The  B.  lepra  stains  quickly; 
the  B.  Smegmatis  is  decolorized 
by  the  alcohol. 

5.  Pappenheim's  Method.     (Supposed  to 

differentiate    between    B.  tubercu- 
losis   and    other    acid-fast  micro- 
organisms), 
(a)  Prepare  films  as  usual. 


BACTERIOLOGY. 


(b)  Stain  in  carbol-fuchsin  without 
heat  for  3  minutes. 

(c)  Without  washing  in  water  treat 
the  film  with  3  or  4  successive 
applications  of  Pappenheim's 
(corallin)  stain. 

(d)  Wash  in  water. 

(e)  Dry  and  mount. 

Neisser's  Method  (for  B.  diphtheria). 

(a)  Prepare  films  as  usual. 

(b)  Treat  with  solution  I,  (Neisser's 
stain)  for  1  to  3  seconds. 

(c)  Wash  in  water. 

(d)  Treat  with  solution  II,  (Neis- 
ser's stain)  for  3  to  5  seconds. 

(e)  Wash,  dry  and  mount. 

By  this  method  the  body  of  the  or- 
ganism is  stained  brown  and  the 
oval  polar  granules  are  blue. 
Modified   Neisser's   Method    (for  B. 
diphtheria). 

(a)  Prepare  films  as  usual. 

(b)  Treat  with  solution  I,  (Modified 
Neisser's  stain)  for  2  minutes. 

(c)  Wash  in  water. 

(d)  Treat  with  Gram's  iodine  solu- 
tion for  10  seconds. 

(e)  Wash  in  water. 

(f)  Treat  with  solution  II,  (Modified 
Neisser's  stain)  for  30  seconds. 

(g)  Wash,  dry  and  mount. 

This  must  be  used  on  cultivations 
grown  upon  blood  serum,  incu- 
bated at  37°  C.  for  from  9  to  1« 
hours. 

The  body  of  the  organism  is  stained 
a  light  red  and  the  granules  are 
black. 

Hunt's  Method  (for  diphtheria). 

(a)  Prepare  films  as  usual. 

(b)  Treat  with  aqueous  methylene- 
blue  for  1  minute. 

(c)  Wash  in  water  and  dry. 

(d)  Treat  with  tannic  acid  (10%  so- 
lution) for  1  minute. 

(e)  Wash  in  water  and  dry. 

(f)  Treat  with  an  aqueous  solution 
of  methyl-orange  for  1  minute. 

(g)  Wash,  dry  and  mount. 

Grain's  Method  with  addition  of  Bis- 
marck-brown  (for  gonococcus). 

(a)  Prepare  film  with  the  urethral 
pus  and  fix. 

(b)  Treat  with  aniline  gentian  violet; 
stain  for  15  seconds. 

(c)  Wash  in  water. 


BACTERIOLOGY. 


81 


(d)  Treat  with  Gram's  iodin  solution 
and  permit  to  remain  for  from 
1   to   2  minutes. 

(e)  Wash  specimen  in  70  per  cent 
alcohol  until  but  a  faint  violet 
color  remains. 

(f)  Stain  for  2  minutes  with  sta.  ale. 
sol.  of  Bismarck-brown. 

(g")  Wash  in  water,  dry  and  mount 
in  balsam. 
By  this  method  the  gentian  violet 
stains  all  bacteria  present,  but 
the  treatment  with  the  iodine 
solution  and  alcohol  decolorizes 
the  gonococcus,  while  the  other 
bacteria  in  the  urethra  remain 
violet. 

The  addition  of  the  Bismarck- 
brown  stains  the  previously  de- 
colorized gonococcus  a  light 
brown.  Nuclei  of  pus  and 
epithelial  cells  are  stained  a  ma- 
hogany color,  while  the  bodies 
of  cells  are  somewhat  lighter  in 
color. 

10.  Wheal  and  Chown  (Oxford)  Method 

(for  actinomyces). 

(a)  Prepare  films  as  usual. 

(b)  Stain  with  Ehrlich's  haema- 
toxylin  till  nuclei  are  a  faint 
blue  after  washing  with  tap 
water  (examine  with  micro- 
scope). 

(c)  Stain  in  hot  carbol-fuchsin  for 
5  to  10  minutes. 

(d)  Wash  in  tap  water. 

(e)  Decolorize  in  alcohol. 

(f)  Dehydrate  in  alcohol. 

(g)  Clear  in  xylol. 

(h)  Mount  in  xylol  balsam. 

Can  also  be  used  for  sections. 
TECHNIQUE   FOB   CAFSUXiE  STAIZT- 
ING. 

1.  John's  Method. 

(a)  Prepare  films  as  usual. 

(b)  Warm  in  a  2%  solution  of  gen- 
tian  violet   till   steam  arises. 

(c)  Wash,  dry  and  mount. 

2.  Welch's  Method. 

(a)  Prepare  films  as  usual. 

(b)  Flood  with  acetic  acid  (2%)  for 
2  minutes. 

(c)  Remove  acetic  acid  by  means  of 
filter  paper  or  blow  it  oft  with 
a  pipette. 


82  BACTERIOLOGY. 


(d)  Treat  with  aniline  gentian  violet 
for  5  to  30  seconds. 

(e)  Wash,  dry  and  mount. 

3.  Hiss'  Method. 

(a>  Prepare  films  as  usual. 

(b)  Treat  with  a  mixture  of  gentian 
violet  or  fuchsin  (5  cc.)  and  dis- 
tilled water  (95  cc.)  heated  until 
it  steams. 

(c)  Wash  in  a  solution  (20%)  of 
cupric  sulphate  crystals. 

(d).  Wash,  dry  and  mount. 

4.  Ritotoert's  Method. 

(a)  Prepare  film  as  usual. 

(b)  Treat  with  Ribbert's  stain  for  1 
to  2  seconds. 

(c)  Wash,  dry  and  mount. 

5.  MacConkey's  Method. 

(a)  Prepare  film  as  usual. 

(b)  Treat  with  MacConkey's  stain  for 
5   to  10  minutes. 

(c)  Wash  thoroughly,  dry  and  mount. 

6.  Muir's  Method. 

(a)  Prepare  film  as  usual. 

(b)  Treat  with  carbol-fuchsin,  warm 
until  steam  begins  to  rise  and 
allow  stain  to  act  for  30  seconds. 

(c)  Wash  ^  quickly  with  methylated 
spirit. 

(d)  Wash  in  water. 

(e)  Treat  with  Muir's  mordant  for 
5  seconds. 

(f)  Wash  in  water. 

(g)  Treat  with  methylated  spirit  for 
30  seconds  (film  should  now  be 
a  pale  red). 

(h)  Wash  in  water. 

(i)  Stain  with  aqueous  solution  of 
methylene-blue  for  30  seconds. 

(j)  Wash  in  water. 

(k)  Dehydrate  in  alcohol. 

(1)  Clear  in  xylol  and  mount. 

TECHNIQUE  FOB  SPORE  STAINING. 

1.  Sing'le  Stain. 

(a)  Prepare  film  as  usual,  except  that 
you  pass  film  through  the  flame 
15  or  30  times  instead  of  the 
usual  three.  This  will  destroy 
resisting  power  of  spore  mem- 
brane and  permits  the  stain  to 
reach  the  interior. 

(b)  Stain  with  methylene-blue  or 
fuchsin. 

(c)  Wash,  dry  and  mount. 

2.  Double  Stain. 


BACTERIOLOGY. 


83 


(a)  Prepare  film  as  usual  (flame  3 
times). 

(b)  Treat  with  carbol-fuchsin,  steam- 
ing for   20  minutes. 

(c)  Wash  in  water. 

(a)  Decolorize  in  acid  alcohol  (97  cc, 
70%  alcohol  and  3  cc.  hydro- 
chloric acid)  for  a  few  seconds, 
in  2  part  alcohol  and  1  part  of 
1%  acetic  acid,  or  in  1%  sulphuric 
acid. 

(e)  Wash  in  water. 

(f)  Examine  under  the  1/6  objective, 
(film  mounted  in  water). 

The  spores  should  be  red  and  the 
rd^s  unstained  or  faintly  pink. 

(g)  Counter  stain  with  weak  methy- 
lene-blue  for  3  to  4  minutes  or 
g-entian  violet  for  1  minute. 

(h)  Wash,  dry  and  mount. 

3.  Abbott's  Method. 

(a)  Prepare  film  as  usual. 

(b)  Treat  with  Loeffler's  alkaline 
methylene-blue,  heat  carefully  till 
steam  arises  and  allow  hot  ^  stain 
to  act  for  1  to  5  minutes. 

(c)  Wash  in  water. 

(d)  Decolorize  in  a  solution  made  up 
of  1  part  of  2%  nitric  acid  and 
98  parts  of  80%  alcohol. 

(e)  Wash  in  water. 

(f)  Counter  stain  in  eosin  (1%  aq.  * 
sol.) 

(g)  Wash,  dry  and  mount. 

4.  Mueller's  Method. 

(a)  Prepare  film  as  usual. 

(b)  Treat  with  absolute  alcohol  for 
2  minutes,  then  in  chloroform  for 
2  minutes.  (This  dissolves  out 
any  fat  or  crystals  that  might 
retain  the  spore  stain). 

(c)  Wash  in  water. 

(d)  Treat  with  a  5%  aqueous  solu- 
tion of  chromic  acid  for  1  minute. 

(e)  Wash  in  water. 

(f)  Decolorize  in  5%  aqueous  solution 
of  sulphuric  acid  for  5  seconds. 

(g)  Wash  in  water. 

(h)  Counter  stain  with  Kuehne's  car- 
bolic methylene-blue  for  1  to  2 
minutes. 

(i)  Wash,  dry  and  mount. 
TECHNIQUE  FOR  FZiAGEUA  STAIN- 
ING. 

Bacteria    shouM    be    from    smear  agar 
cultures,  12  to  18  hours  old  if  incu- 


84  BACTERIOLOGY. 


bated  at  37°  C,  24  to  30  hours  if 
incubated  at  22°  C. 

In  preparing  the  films  a  small  quantity 
of  the  growth  is  removed  by  means 
of  the  platinum  loop  and  transferred 
to  a  few  cc.  of  distilled  water  in  a 
watch  glass.  Gently  mix  the  bacteria 
with  the  water  by  moving  the  loop  to 
and   fro,   without   touching   the  side 

.  of  the  watch-glass.  Flame  a  cover 
slip  and  spread  a  thin  film,  using  no 
force.  Dry  in  air,  protect  the  film 
from  dust.  Hold  the  cover  slip  be- 
tween finger  and  thumb,  and  fix  by 
passing  3  times  through  the  flame. 

1.  Xioefder's  Metliod. 

(a)  Prepare  film  as  described  above. 

(b)  Treat  with  Loeflfler's  mordant, 
hold  it  high  above  the  flame  and 
heat,  steaming  for  1  minute. 

(c)  Wash  in  water  (distilled)  and  dip 
carefully  in  absolute  alcohol. 
Wash  again  in  water. 

(d)  Filter  on  to  the  film  a  few  drops 
'  of  Loefiler's   "flagella  stain"  and 

warm  as  before  for  1  minute. 

(e)  Wash,  dry  and  mount. 

2.  Bungfe's  Method. 

Same  as  Loeffier's,  except,  that 
Bunge's  mordant  is  substituted 
for  Loeflaer's. 

3.  Pitfield's  Method. 

(a)  Prepare  film  as  described  above. 

(b)  Mix  equal  parts  of  Pitfield's 
mordant  and  stain,  boil  the  mix- 
ture, and  while  still,  hot  immerse 
the  film  in  it  for  1  minute. 

(c)  Wash  in  water. 

(d)  Examine  in  water;  if  satisfactory, 
dry  and  mount. 

4.  Muir's  Modined  Fitiield  Method. 

(a)  Prepare  film  as  described  above. 

(b)  Treat  with  Muir's  mordant,  hold 
it  high  above  the  fiame  and  heat, 
steaming  for  2  minutes. 

(c)  Wash  in  water  and  dry  carefully. 

(d)  Treat  with  Muir's  stain  (for 
flagella)  and  warm  as  before  for 
2  minutes. 

(e)  Wash  carefully,  dry  and  mount. 

5.  Van  Ermengrem^s  Method. 

(a)  Prepare  film  as  described  above. 

(b)  Treat  with  Van  Ermengem's  fix- 
ing solution,  heat  carefully, 
steaming  for  5  minutes, 

(c)  Wash  in  water. 


BACTERIOLOGY. 


85 


(d)  Wash 0  in  absolute  alcohol. 

(e)  Wash  in  distilled  water. 

(f)  Treat  with  Van  Ermengem's 
"sensitizing  solution"  for  to  1 
minute;  remove  excess  of  fluid 
with  filter  paper. 

(g)  Transfer  film  to  a  watch-glass 
containing  Van  Ermengem's  "re- 
ducing solution"  for  %  to  1  min- 
ute; remove  excess  of  fluid  with 
filter  paper. 

(h)  Treat  again  with  the  "sensitizing 
solution"  until  film  commences  to 
turn  black. 

(i)  Wash  in  distilled  water,  dry  and 
mount. 

TECHNIQUE  FOB  STAINING  BAC- 
TERIA IN  TISSUES. 

This  is '  practically  the  same  as  prepar- 
ing tissue  for  histological  study. 
Small  pieces  of  tissue  are  selected 
and 

Fixed  in  alcohol  (most  used;  formalin, 
Zenker's  fluid  or  Mueller's  fluid 
are  also  used  but  are  not  so  good 
as  the  alcohol  fixative). 

Hardened,  unless  alcohol  is  used  as 
fixative;  if  not,  then  the  tissues 
must  be  kept  for  24  hours  in  50%, 
75%,  90%  and  absolute  alcohols. 

Dehydrated,  by  transferring  the  tis- 
sues to  fresh  absolute  alcohol. 

Cleared  by  xylol  or  chloroform. 

Embedded  in  paraflan,  (Ceiloidin  Is 
also  used  but  is  not  pref eralple). 

iSectioued.  Sections  are  fioated  on 
slide  which  has  been  lightly 
smeared  with  a  mixture  of  equal 
parts  egg  albumin  and  glycerine  i 
to  which  is  added  a  crystal  of 
camphor  or  a  drop  or  two  of 
carbolic  acid.  It  is  now  put 
;aside  in  the  incubator  (or  warm- 
ing chamber)  for  4  or  5  hours. 

Stained  Iby 

XoeiHer's  Method. 

1.  Dissolve  out  paraffin  with  xylol. 

2.  Remove  xylol  by  flushing  section 

with  absolute  alcohol. 

3.  Stain    in    alcoholic  methylene-blue 

solution  for  5  to  15  minutes,  or 
in  Loeffier's  alkaline  methylene 
blue  for  from  1  to  24  hours. 

4.  Wash  in  1-1000  solution  of  acetic 

acid  for  about  10  seconds. 


86 


BACTERIOLOGY. 


5.  Treat    with    absolute    alcohol  for 

10-20  seconds. 

6.  Clear  in  xylol. 

7.  Mount  In  balsam. 
Gram-Weig-ert  Metliod.    (To  stain  Gram 

positive  bacterial). 

1.  Dissolve  out  paraffin  with  xylol. 

2.  Remove  xylol  by  flushing  section 

with  absolute  alcohol. 

3.  Stain  in  alum  carmin  for  about  15 

minutes. 

4.  Wash  thoroughly  in  water. 

5.  Filter  aniline  gentian  violet  solu- 

tion on  to  the  section  and  allow 
to  stain  for  about  25  minutes. 

6.  Wash  thoroughly  in  water. 

7.  Treat    with    Lingol's    iodine  until 

section  ceases  to  become  any 
blacker. 

8.  Wash  thoroughly  in  water. 

9.  Treat  with  a  mixture  of  equal  parts 

of  aniline  oil  and  xylol  until  no 
more  color  comes  away. 

10.  Wash  thoroughly  with-xylol. 

11.  Decolorize  and  dehydrate  with  ab- 

solute alcohol  until  there  remains 
only  a  very  faint  bluish  tint. 

12.  Clear  with  xylol. 
1^.  Mount  in  balsam. 

The  fibrin  and  hyaline  tissue  are 
stained    deep    blue    while  Gram 
positive   bacteria   appear   a  deep 
blue  violet  color. 
To  Stain  Acid  fast  Bacteria. 

1.  Prepare    sections    for    staining  as 

above. 

2.  Stain  with  haematin  solution  10  to 

20  seconds,  to  obtain  a  pure 
nuclear  stain. 

3.  Wash  in  water. 

4.  Stain  with  carbol  fuchsin  for  from 

20  to  30  minutes  at  47°  C. 

5.  Wash  in  water. 

6.  Treat   with   aniline  hydrochlorate, 

2%  watery  solution,  for  from  2  to 
5  seconds. 

7.  Decolorize  i'n  75%  alcohol  till  sec- 

tion appears  free  from  stain  (15 
to  30  minutes). 

8.  Dehydrate  with  absolute  alcohol. 

9.  Clear  with  xylol. 
10.  Mount  in  balsam. 

To  Stain  Actinomyces. 
Mallory's  Method. 

1.  Prepare    sections    for    staining  as 
above. 


BACTERIOLOGY. 


87 


2.  Stain  with  a  saturated  watery  solu- 

tion of  eosin  for  10  minutes. 

3.  Wash  in  water. 

4.  Apply    aniline    gentian    violet  for 

from  2  to  5  minutes. 

5.  Wash  in  normal  saline  solution'. 

6.  Apply    Weig^ert's    iodine  solution 

(Iodine  1  part,  K.  I.  2  parts  and 
water  100  parts)  for  1  minute. 

7.  Wash  in  water  and  blot. 

8.  Clear  in  aniline  oil. 

9.  Wash  in  several  changes  of  xylol. 
10.  Mount  in  balsam. 


STUDY  OF  CHEMICAL  PRODUCTS  OF 
GROWTH  (BIOCHEMICAL  METHODS). 

Effect  of  Physical  Ag-ents  on  Growth 
Study  of  Disinfectants. 

1.  TEST    POB    THE    PRESENCE  OP 
ENZYME  PRODUCTION. 

(a)  Proteolytic  by 

Preparing  cultivations  in  flask 
bulk  (50  cc),  using  blood  serum 
and  milk  serum  filtered  through 
porcelain;  incubate;  after  which 
the  liquid  is  made  faintly  acid 
(acetic  acid  1%)  and  boiled;  a 
precipitate  of  unaltered  proteins 
is  thrown  down.  Filter.  Mix 
10  cc,  of  the  filtrate  and  1  cc. 
of  caustic  soda  (30%)  in  a  test 
tube;  add,  drop  by  drop,  of 
copper  sulphate  solution  (0.5%). 
A  pink  color  which  becomes 
violet  as  copper  sulphate  is 
added  =  proteose  and  peptone. 
Saturate  the  rest  of  filtrate  with 
ammonium  sulphate.  The  pre- 
cipitate =  proteose. 

Filter  and  divide  the  filtrate  into 
3  parts. 

(1)  In  one  part  use  excess  of 
caustic  soda  (30%  aq.  sol.)  to 
displace  the  ammonia  from  the 
ammonium  sulphate,  then  add 
drop  by  drop  of  the  copper  sul- 
phate solution  (0.5%),  —  a  pink 
color  =  peptone. 

(2)  Boil  second  part  with  Millon's 
reagent  (a  solution  of  mer- 
curic nitrate  in  water  contain- 
ing free  nitrous  acid,  —  a  pink 
color  =  peptone. 


BACTERIOLOGY. 


(3)  Add  to  the  3rd  part  some 
glyoxylic  acid  solution,  then  run 
in  sulphuric  acid  (cong),  — 

A  violet  ring  at  upper  level  of 
acid  =:  tryptophane. 

(b)  Diastase,    by    preparing  inosite- 

free  bouillon  tube  cultivations 
and  incubate.  Add  equal  parts 
of  the  cultivations  and  a  thin 
starch  paste  (made  with  water 
and  starch  to  which  is  added 
2%  of  thymol);  incubate  the 
mixture  and  incubate  at  37°  C. 
for  6  to  8  hours.  Filter.  Test 
the  filltrate  for  sugar,  using 
"Fehlings  test" — a  yellow  or 
orange  precipitate  —  sugar. 

(c)  Invertase,   by   preparing  inosite- 

free  bouillon  tube  cultivations 
and  incubate.  Mix  equal  parts 
of  the  cultivation  and  carbolized 
sugar  solution  (carbolic  acid  2 
parts,  cane  sugar  2  parts  and 
water  96  parts)  in  a  test  tube; 
allow  to  stand  for  several 
hours.  Filter.  Test  the  filtrate 
as  in  the  Diastase. 

(d)  Reunin,  by  preparing  inosite-free 

bouillon  tube  cultivations  and 
incubate.  Heat  the  cultivation 
to  55°  C.  for  30  minutes  (to 
sterilize).  With  a  sterile  pi- 
pette run  5  cc.  of  the  cultivation 
into  each  of  3  tubes  of  litmus 
milk.  Incubate  at  22°  C,  and 
examine  each  day  for  10  days. 

Absence  of  coagulation  =  absence 
of  rennin  ferment. 

Fermentation  Reactions  are  made 
upon  peptone  water  containing 
2%  respectively  of  each  of  the 
following: — a  monosaccharide 
(dextrose),  disaccharide  (lac- 
tose), trisaccharide  (mellitose), 
polysaccharide  (dextrin)  and 
glucocide  (amygdalin) ;  also 
1%  respectively  of  each  of  the 
following  organic  salts: — so- 
dium citrate,  formate,  lactate, 
maltate  and  tartrate.  Make  tube 
cultivations  in  each  of  the 
above,  observe  from  day  to  day 
for  10  days  and  note  growth, 
reaction,  gas  production. 


BACTERIOLOGY.  89 

2.  TEST    FOB    THE    PRESENCE  OF 
ACID  PRODUCTION. 

(a)  Quantitative..     Prepare  glucose 

bouillon  cultivation  in  bulk 
(100  cc.)  in  a  flask;  also  "con- 
trol" flask  of  medium.  Incubate 
both  flasks.  Heat  in  Arnold  for 
30  minutes  to  sterilize.  De- 
termine the  titre  of  the  "inoc- 
ulated'.' and  "control"  medium; 
the  difference  between  the  titre 
gives  the  total  acid  production 
of  the  bacterium  in  terms  of 
normal  NaOH. 

(b)  Qualitative.     Prepare  glucose  or 

lactose  bouillon  cultivation,  in 
bulk  (500  cc),  in  a  litre  flask 
and  add  10  gms.  of  sterilized 
precipitated  chalk.  Incubate. 
Put  a  cube  (about  1  cc.)  of 
paraffin  into  the  cultivation  and 
connect  it  up  with  a  condenser. 
Distill  over  200  to  300  cc.  This 
distillate  (1st  distillate  "A") 
will  contain  alcohol,  etc.,  (see 
Alcohol  production).  The  first 
residue  "a,"  will  contain  the  vol- 
atile and  fixed  acids.  Filter 
the  first  residue  ("a")  and  make 
up  the  filtrate  (first  filtrate  "a") 
with  distilled  water  to  500  cc. 
and  divide  into  2  parts.  Treat 
250  cc.  (1st  portion  of  filtrate 
"a")  with  20  cc.  phosphoric  acid 
(cong.)  to  liberate  the  volatile 
acids  and  distill  (second  dis- 
tillate "B")  to  small  bulk.  The 
second  distillate  ("B"),  may- 
contain  formic,  acetic,  propionic, 
butyric  and  benzoic  acids.  Add 
baryta  water  till  alkaline  and 
evaporate  to  dryness.  Add  50  cc. 
absolute  alcohol,  allow  to  stand, 
stirring  frequently,  for  2  or  3 
hours. 

Filter  and  wash  with  alcohol. 

Filtrate  (2nd  "b")  may  contain 
barium  propionate,  barium  bu- 
tyrate.  Evaporate  to  dryness 
and  dissolve  (2nd  filtrate  "b") 
with  150  cc.  water.  Acidify 
with  phosphoric  acid  and  dis- 
till (2nd  "b").  Saturate  the 
distillate  with  calcium  chloride 
and  distill  over  a  few  cc.  — 
(third  distillate  "c").    Test  dis- 


BACTERIOLOGY. 


tillate  for  butyric  acid  (add 
3  cc.  alcohol  and  4  drops  sul- 
phuric acid  cong.  Smell  of  pine- 
apple— butyric  acid). 
Besidue  (3rd  "C")  may  contain 
barium  acetate,  barium  formate, 
barium  benzoate.  Evaporate  off 
alcohol  and  dissolve  up  residue 
on  filter  in  hot  water  and  neu- 
tralize. 

Divide  the  solution  into  4  por- 
tions. 

(1)  Add  ferric  chloride  solution 
(4%  aq.).  Brown  color  =  acetic 
or  formic  acids;  buff  ppt.  — 
benzoic  acid.  (2)  Add  silver  ni- 
trate solution  (1%  aq.),  then  1 
drop  ammonia  water  and  boil; 
black  ppt.  of  metallic  silver  = 
formic  acid.  (3)  Evaporate  to 
dryness;  mix  with  equal  quanti- 
ties of  arsenius  oxide  and  heat 
on  platinum  foil;  unpleasant 
smell  of  cacodyl  =  acetic  acid. 
(4)  Add  a  few  drops  of  mer- 
curic chloride  solution  in  a  test 
tube,  and  heat  to  70°  C,  pre- 
cipitate of  mercurous  chloride 
which  is  slowly  reduced  to 
mecury  =  formic  acid. 

Second  residue  ("b").  Wash  from 
filter  paper,  dissolve  by  heating 
with  dilute  hydrochloric  acid 
(25%)  and  add  calcium  chloride 
solution  and  ammonia  until  al- 
kaline. 

White    precipitate    insoluble  in 

acetic  acid  —  oxalic  acid. 
2iLd  Portion  of  first  filtrate  C<a") 

(2nd  250  cc.)  should  be  ex- 
amined for  (the  ether  soluble 
acids)  lactic,  oxalic,  succinic, 
benzoic;  salicylic,  gallic  and 
tannic  acids  by  evaporating  the 
filtrate  to  a  thin  syrup,  acidify 
strongly  with  phosphoric  acid. 
Extract  by  agitation  in  a  sepa- 
ratory  funnel  with  5  times  its 
volume  of  ether.  Evaporate  the 
ethereal  extract  to  a  thin  syrup. 
Add  100  cc.  of  water  and  mix. 
Add  a  slight  excess  of  sodium 
carbonate  to  a  small  portion  of 
the  mixture  and  evaporate  to 
dryness  on  a  water-bath,  dis- 
solve in  5  to  10  cc.  of  pure  sul- 


BACTERIOLOGY. 


91 


phuric  acid,  add  2  drops  of 
copper  sulphate  (sat.  sol.)  place 
in  a  test  tube  and  heat  in  boil- 
ing water-bath  for  2  minutes; 
cool,  add  2  or  3  drops  of  thio- 
phene  solution  (0.15  cc.  in  100  cc. 
alcohol)  and  warm  gently. 
Cherry  red  color  =  lactic  acid. 
If,  on  the  addition  of  the  sul- 
phuric acid,  a  brown  color  is 
produced,  another  sample  should 

.  be  taken  and  boiled  with  animal 
charcoal  before  evaporating. 

If  lactic  acid  is  present,  prepare 
zinc  lactate  by  boiling  part  of 
the  solution  of  the  ether  extract 
with  excess  of  zinc  carbonate, 
filtering  and  evaporating  to 
crystalize.  The  crystals  ob- 
tained have  a  characteristic 
form,  and  it  dried  at  110°  C, 
should  contain  26.87%  of  zinc. 

Test  a  part  of  the  rest  of  the  so- 
lution of  the  ether  extract  for 
oxalic  acid  by  heating  with  di- 
lute hydrochloric  acid  and  add- 
ing calcium  chloride  solution 
and  ammonia  until  alkaline. 
White  precipitate  insoluble  in 
acid  =:  oxalic  acid.  Neutralize 
the  remainder  and  add  ferric 
chloride  solution  (4%  aq.  sol.). 
Red  brown  gelatinous  precipi- 
tate =  succinic  acid.  Buff  pre- 
cipitate =  benzoic  acid  and 
other  acids  related  to  benzoic 
acid.  Violet  color  =  salicylic 
acid.  Inky  black  color  precipi- 
tate =:  gallic  or  tannic  acid. 

TEST    FOB    THE    PRESENCE  OF 
AMMONIA  PBODT7CTION. 

Prepare  cultivation  in  bulk  (100 
cc.)  in  a  250  cc.  flask  and  in- 
cubate together  with  a  control 
flask. 

After  incubation  add  2  gms.  of 
calcined  magnesia  to  each  flask, 
then  connect  up  with  con- 
densors  and  distill.  Place  50  cc. 
of  the  distillate  from  each  in  a 
Nessler  tube  and  add  1  cc.  16 
gms.  Hgclz  in  500  cc.  pure  water, 
35  gm.  K.  I.  in  200  cc.  pure 
water.  Pour  Hgclg  solution  into 
K.  I.  solution  until  faint  show 
of  excess  is    indicated.    Add  160 


92 


BACTERIOLOGY. 


gms.  KOH.  Dilute  to  1000  cc 
and  add  a  strong  solution  of 
Hgcls  until  red  mercuric  iodide 
just  begins  to  be  prominent. 
Let  excess  of  mercuric  iodide 
settle  to  bottom.  Reagent  should 
have  a  pale  straw  color  to  each 
tube.  Yellow  color  =  ammonia. 
The  depth  of  color  is  propor- 
tionate to  the  amount  present. 

4.  TEST  FOB  THE  FBESENCi:  OF  A3j- 
COHOIklC  FBODUCTION. 

Prepare  glucose  or  lactose  bouil- 
lon cultivation  in  bulk  (500  cc.) 
in  a  litre  flask  and  add  10  gms. 
of  sterilized  precipitated  chalk. 
Incubate.  Put  a  cube  (about 
1  cc.)  of  paraffin  into  the  culti- 
vation and  connect  it  up  with 
a  condenser.  Distill  over  200 
or  300  cc.  'Divide  the  distillate 
into  4  portions  and  test  for  the 
production  of  alcohol,  acetalde- 
hyde,  acetone.  Add  Lugol's 
Iodine,  then  a  little  NaOH  so- 
lution and  stir  till  the  color  of 
the  iodine  disappears.  Pale- 
yellow  crystalline  precipitate  of 
iodoform  (with  characteristic 
smell)  appearing  in  the  cold,  = 
acetaldehyde,  or  acetone;  ap- 
pearing only  on  warming  =  al- 
cohol. The  precipitate  may  be 
absent,  .  though  odor  is  pro- 
nounced' Add  Schiff's  reagent 
(sulphuric  acid  3  parts  and  a 
10%  solution  of  ferricchloride, 
1  part).  Violet  or  red  color  = 
aldehyde. 

Add  to  10  cc.  of  the  solution,  2.5 
cc.  of  a  25%  sulphuric  acid,  and 
a  crystal  or  two  of  potassium 
bichromate  and  distill.  Reduc- 
tion of  the  bichromate  to  a 
green  color  and  a  distillate, 
which  smells  of  acetaldehyde 
and  reacts  with  Schilf's  reagent 
=  presence  of  alcohol  in  the 
original  liquid.  Add  a  few  drops 
of  sodium  nitroprusside  liquid 
solution  (5%),  add  ammonia  to 
make  alkaline,  then  saturate 
with  ammonium  sulphate  crys- 
tals. Acetone  gives  little  color 
on  the  addition  of  ammonia,  but 
after  the  addition  of  ammonium 


BACTERIOLOGY. 


93 


sulphate  a  deep  permanganate 
color  is  found,  which  takes  10 
minutes  to  reach  its  full  depth. 
Aldehyde  gives  a  carmine  red  not 
changed  by  the  ammonium  sul- 
phate. 

5.  TEST  rOR  THIS  PRODUCTION  OF 
INDOIi,  (a  product  of  putrefaction). 
Make  several  peptone  water  test 
tube  cultivations  and  incubate. 
Allow  the  culture  to  cool  to 
room  temperature  and  remove 
2  cc.  of  the  cultivation  by  means 
of  a  sterile  pipette,  transfer  to 
clean  tubes  then  add  2  cc.  para- 
dimethylamino-benzaldehyde  so- 
lution (paradimethylamino-ben- 
zaldehyde  4  gms.,  absolute  alco- 
hol 380  cc.)  add  2  cc.  potassium 
persulphate  solution  (sat.  aq. 
sol.).  A  delicate  rose-pink  color 
appearing  throughout  the  mix- 
ture which  deepens  slightly  on 
standing  indicates  indol. 
A  method  used  for  the  test  in 
several  laboratories  is  by  the 
ordinary  nitrosoindol  reaction. 
Inoculate  several  tubes,  each 
containing  10  cc.  of  glucose-free 
bouillon  or  peptone  water,  allow 
the  culture  to  grow  for  5  to  10 
days.  To  each  tube  of  the  cul- 
ture add  10  drops  of  pure  con- 
centrated sulphuric  acid,  and 
then  1  cc.  of  a  sodium  nitrate 
solution  (.2%).  If  a  pink  color 
develops  within  10  minutes,  in- 
dol is  present.  In  recording  the 
production  of  indol  it  is  neces- 
sary to  state  the  age  of  the  cul- 
ture, since  indol  may  be  pro- 
duced in  10  days  and  not  in  five 
days. 

The  reaction  may  appear  immedi- 
ately, or  a  faint  reaction  may 
appear  after  a  long  standing. 

The  test  can  also  be  performed  in 
one  stage  by  making  a  mixture 
of  concentrated  commercial  sul- 
phuric, hydrochloric  or  nitric 
acid,  all  of  which  hold  a  trace 
of  nitrite  in  solution.  A  red 
color  within  20  minutes  pres- 
ence of  indol. 


94  BACTERIOLOGY. 

6.  TEST  FOR  THE  PBODUCTION  OF 
FHENOIi  (a  product  of  putrefac- 
tion). 

Prepare  a  50  cc.  nutrient  bouillon 
cultivation  in  a  100  cc.  Erlen- 
meyer  flask  and  incubate.  After 
incubation  add  5  cc.  of  sul- 
phuric acid  solution  (25%), 
connect  up  with  a  condenser, 
distil  over  15  to  20  cc.  Divide 
the  distillate  into  3  portions. 
To  one  portion  add  0.5  cc.  of 
Millon's  reagent.  Digest  one 
part  by  weight,  of  mercuric 
chloride  with  2  partso  by  weight, 
of  nitric  acid  sp.  gr.  1.42  and 
dilute  the  resulting  solution 
with  2  volumns  of  water  and 
heat  to  boiling.  Red  color 
phenol. 

To  another  portion  add  0.5  cc.  of 
a  ferric  chloride  solution  (1% 
aq.  sol.).  Violet  color  =  phenol. 
(If  the  distillate  is  acid  the  re- 
sult will  be  negative). 

To  another  portion  add  strong 
bromine  water. 

A  crystalline  white  ppt.  of  tri- 
bromo-phenol  or  a  turbidity  = 
phenol. 

In  recording  the  presence  or  ab- 
sence of  phenol  in  cultures,  the 
age  of  the  culture  and  tempera- 
ture of  growth  should  be  stated. 
If  indol  and  phenol  appear  to- 
gether in  the  same  culture,  it  is 
well  to  separate  them  before 
making  the  tests,  by  the  Hoppe- 
Deyler  method,  in  the  following 
manner:  Make  a  200  to  300  cc. 
mosite-free  bouillon  flask  culti- 
vation and  incubate,  after  which, 
render  deflnitely  acid  with 
acetic  acid  and  connect  up  with 
a  condenser.  Distill  over  50  to 
70  cc.  The  distillate  will  con- 
tain both  indol  and  phenol. 
Render  the  distillate  strongly 
alkaline  with  caustic  potash  and 
redistill.  The  distillate  will 
contain  indol  (make  test  for 
same)  while  the  residue  will 
contain  phenol.  When  the  resi- 
due is  cold,  saturate  it  with 
carbon  dioxide  and  redistill. 
Test  this  distillate  for  phenol. 


BACTERIOLOGY.  95 

7.  TEST  FOR  THE  PRODUCTION  OF 

REDUCING  AGENTS. 

Color  destruction.  Prepare  tube 
cultivations  in  nutrient  bouil- 
lon faintly  colored  with  litmus, 
rosolic  acid,  neutral  red,  and  in- 
cubate. Examine  the  cultures 
from  day  to  day  and  note 
whether  any  color  change  oc- 
curs. 

Reduction  of  Nitrates  to  Nitrites. 

Prepare  tube  cultivations  of  ni- 
trate bouillon  on  nitrate  peptone 
and  incubate  together  with  un- 
inoculated  controle  tubes.  This 
is  necessary  as  the  medium 
may  take  up  nitrates  from  the 
atmosphere  and  an  opinion  as 
to  the  absence  of  nitrates  in 
the  cultivation  is  based  upon  an 
equal  coloration  of  the  medium 
in  the  controle  tube.  Test  both 
the  culture  and  the  control  for 
the  presence  of  nitrates  by  add- 
ing drops  of  sulphuric  acid 
(25%)  to  the  tubes,  then  run  in 
2  or  3  cc.  metaphenylenediamine 
(5%  aq.  sol.)  into  each  tube. 
Brownish-red  color  =  nitrites. 
The  depth  of  color  is  in  pro- 
portion to  the  amount  present. 

8.  TEST  FOR  THE  PRODUCTION  OF 

PIGMENTS. 

Make  tube  inoculations  upon  the 
various  media  and  incubate  at 
37°  and  20**  C.  temperatures; 
grow  them  also  under  aerobic 
and  anaerobic  atmospheres; 
also,  exposed  to  and  protected 
from  light.  Note  the  conditions 
most  favorable  to  the  formation 
pigments.  Note  the  solubility 
of  the  pigment  in  hot  and  cold 
water,  alcohol,  ether,  chloro- 
form, benzol  and  carbonbisulph- 
ide.  Note  the  effect  of  acids  and 
alkalies  upon  the  cultivations  or 
upon  solutions  of  the  pigment. 

9.  TEST  FOR  THE  PRODUCTION  OF 

GAS. 

Inoculate  fermentation  tubes  filled 
with  sugar  bouillons  and  incu- 
bate. Examine  at  intervals  of 
24  hours  and  mark  the  levels 
of  the  fluid  when  the  evolution 
of  gas  has  ceased,  measure  the 


96 


BACTERIOLOGY. 


length  of  the  column  of  gas 
with  a  millimeter  scale.  Ex- 
press this  column  as  a  percent- 
age of  the  entire  length  of  the 
closed  branch. 

To  roughly  determine  the  relative 
proportions  of  CO2  and  H — 
fill  the  bulb  of  the  fermenta- 
tion tube  with  caustic  soda  so- 
lution; close  the  mouth  of  the 
bulb  with  a  rubber  stopper;  in- 
vert the  tube  10  times,  that  the 
soda  may  be  brought  into  in- 
timate contact  with  the  gas; 
return  the  gas  to  the  end  of 
the  closed  tube,  and  measure. 
The  loss  in  volume  of  gas  = 
carbon  dioxide. 

The    residual    gas    =  hydrogen. 
Transfer  the  gas  to  the  bulb, 
apply  a  light  and  cause  it  to 
explode. 
Sulplmretted  Hydrog'ezi. 

Inoculate  tubes  of  iron  peptone 
or  lead  peptone  media  and  in- 
cubate together  with  control 
tubes.  Examine  every  24  hours 
for  several  days.  The  libera- 
tion of  H2  S  will  cause  the 
yellowish-white  precipitate  to 
darken  to  a  brownish-black  or 
jet  black,  the  depth  of  color 
being  in  proportion  to  the 
amount  of  H2  S  present. 
STUBY  OF  THE  BIOI.OGY  OF  CUl^- 
TUBES  BY  FKYSICAI^  METHODS. 

The  growth  and  development  of 
cultures    are    to    be  examined 
under  conditions  of 
1.  Atmosphere.    Prepare  4  sets  of  cul- 
tivation in: — 
(a)  Slanted  glucose  formate  agar  and 
incubate  aerobically  at  37°  C, 
slanted   glucose   formate  gela- 
tine and  incubate  aerobically  at 
20°    C.     Seal    the   cultures  in 
Buchner's    tubes    according  to 
Buchner's  method. 

(c)  Slanted    glucose    formate  agar; 

glucose  formate  bouillon.  Grow 
anaerobically  by  placing  them 
in  Bulloch's  apparatus  accord- 
ing to  Bulloch's  method  and  in- 
cubate at  37°  C. 

(d)  Slanted  glucose  formate  gelatin; 

glucose  formate  bouillon.  Grow 


BACTERIOLOGY. 


97 


anaerobically  by  placing:  them 
in  Bulloch's  apparatus  accord- 
ing to  Bulloch's  method  and 
incubate  at  20°  C. 

Make  observations  upon  the  culti- 
vations both  microscopically  and 
*  microscopically  at  intervals  of 

24  hours,  until  the  completion 
of  7  days  incubation.  By  this 
method  it  can  be  determined 
as  to  whether  an  organism  is 
an  obligate  aerobe,  a  faculta- 
tive anaerobe  or  an  obligate 
anaerobe. 

A  rough  estimate  of  the  above 
may  be  made  by  an  observa- 
tion of  the  growth  in  fermenta- 
tion tubes.  If  there  is  a  growth 
in  the  closed  arm  as  well  as  in 
the  bulb  of  the  tube,  it  indi- 
cates that  the  organism  is  a 
facultative  anaerobe;  while  a 
growth  odburing  only  in  the 
bulb  or  in  the  closed  arm  shows 
that  it  is  an  obligate  aerobe  or 
anaerobe  respectively. 

In  addition  to  the  observation  of 
growth  in  the  presence  or  ab- 
sence of  oxygen  it  is  often 
necessary  to  observe  the  growth 
in — Gases  other  th.au  Ozysrezi 
(SO2,  N2O,  NO,  CO2,  etc.) 

Prepare  tube  cultivations  upon 
solid  media  and  place  them  in 
Bulloch's  apparatus.  Replace 
the  contained  air  with  the 
selected  gas  and  incubate  under 
optimum  temiperature. 

Examine  the  cultivations  at  24 
hour  intervals  for  7  days,  by 
removing  a  tube  from  the  ap- 
paratus each  day. 

If  no  growth  is  visible,  incubate 
the  tube  under  optimum  condi- 
tions of  temperature  and  at- 
mosphere, by  which  the  length 
of  exposure  to  the  gas  neces- 
sary to  kill  the  organism  is  de- 
termined. 
Temperature. 

(a)  Test   the   ran§re   of  temperature 
(minimum  and  maximum). 

Prepare  10  tube  cultivation  in 
fluid  media  of  optimum  reac- 
action.  Arrange  a  series  of  in- 
cubators   with    fixed  tempera- 


BACTERIOLOGY. 


tures  varying  from  ^°  C.  to 
50°  C.  (Water  baths  can  be 
used  if  incubators  are  not 
available).  Incubate  one  tube 
aerobically  and  one  anaerobic- 
ally  in  each  incubator.  Ex- 
amine at  %  hour  intervals  for 
from  5  to  18  hours.'  Note  the 
temperature  (optimum)  at 
which  the  growth  is  first  ob- 
served. 

Continue  the  incubation  for  7 
days.  Note  the  extremes  of 
temperature  at  which  growth 
takes  place  (minimum  and 
maximum  temperatures) 

(b)  Test  for  tlie  (absolute)  optimnm. 

temperature. 
Prepare  10  tube  cultivations  in 
fluid  media  of  optimum  reac- 
tion. Arrange  a  series  of  in- 
cubators w|th  fixed  tempera- 
tures varying  1°  C.  for  5  de- 
grees on  either  side  of  the 
optimum  observed  in  testing 
for  the  range  of  temperature, 
and  incubate  a  tube  in  each 
incubator.  Examine  at  l^  hour 
intervals  and  note  the  tempera- 
ture ("absolute  optimum")  at 
which  growth  is  first  observed. 

(c)  Test  for  the  Thermal  Death-point. 
Veg-etative  Forms. 

Moist  t.  d.  p.  is  the  temperature 
which  kills  a  watery  suspen- 
,  sion  of  the  organism  after  an 
exposure  for  10  minutes. 

Make  the  test  by  preparing  tube 
cultivation  on  solid  media  of 
optimum  reaction,  and  incubate 
for  48  hours.  Examine  cultiva- 
tions microscopically  to  deter- 
mine the  absence  of  spore; 
place  3  loopfuls  of  the  surface 
growth  in  each  of  12  test  tubes 
containing  5  cc.  salt  solution; 
mix  the  organisms  through  the 
salt  solution;  transfer  the  mix- 
ture from  each  tube  into  a 
sterile  250  cc.  flask  and  mix; 
place  5  cc.  of  this  mixture  into 
each  of  12  sterile  test  tubes 
and  number  them  consecutive- 
ly; regulate  a  water  bath  to 
40°  C.  and  suspend  one  tube  in 
it  so  that  the  upper  level  of 


BACTERIOLOGY-. 


99 


the  bacterial  suspension  in  the 
tube  is  about  4  cm.  below  the 
surface  of  the  water  in  the 
bath,  and  the  bottom  of  the 
tube  is  about  the  same  distance 
''rom  the  bottom  of  the  bath; 
suspend  and  adjust  a  "control 
tube"  containing  5  cc.  of  salt 
solution,  under  similar  condi- 
tions, plug  the  tube  with  cot- 
tion  and  pass  a  thermometer 
through  the  plug  so  that  its 
bulb  is  immersed;  watch  the 
themometer  in  the  test  until  it 
records  40"  C.  Note  the  time 
and  at  the  end  of  10  minutes 
at  this  temperature,  remove  the 
bacterial  suspension  and  cool  it 
by  placing  the  lower  end  of  the 
tube  in  running  water.  Make 

3  gelatin  or  agar  plates,  con- 
taining respectively  0.2,  0.3  and 
0.5  cc.  of  the  suspension,  and 
incubate;   place   the  remaining 

4  cc.  of  the  suspension  into  a 
flask  containing  250  cc.  of 
bouillon  and  incubate.  Observe 
the  cultivations  from  day  to 
day  for  7  days,  at  the  end  of 
which  time  "no  growth"  can 
be  recorded. 

The  same  technique  is  to  be  car- 
ried out  in  the  remaining  11 
tubes,  varying  the  conditions 
so  that  each  tube  will  be  ex- 
posed to  a  temperature  2°  C. 
higher  than  the  immediately 
preceding  one  (i.  e.,  42*  C.  44°  C. 
4°6  C.  48"  C,  etc.) 

Note  the  lowest  temperature  at 
which  no  growth  takes  place 
up  to  the  end  of  seven  days'  in- 
cubation, =  the  thermal  death- 
point. 

Dry  t.  d.  p.  is  that  temperature 
which  kills  a  thin  film  of  the 
organism  after  an  exposure  for 
10  minutes. 

Make,  under  sterile  conditions,  an 
emulsion  with  three  loopfuls 
from  an  optimum  cultivation  in 

5  cc.  normal  salt,  and  with  the 
microscope  determine  the  ab- 
sence of  spores.  If  spores  are  ab- 
sent, make  12  cover-slip  sterile 
films  and  place  each  in  a  sterile 


100  BACTERIOLOGY. 


Petri  dish  to  dry.  Expose  each 
dish  in  the  hot-air  oven  for 
ten  minutes  at  temperatures 
varying-  5°  C.  between  60°  C. 
and  120''  C,  with  a  sterile 
forcep  removing-  each  ^Im  from 
the  oven  as  soon  as  the,  ten 
minutes  are  completed,  placing 
it  in  a  flask  containing  200  cc. 
bouillon;  incubate  and  prepare 
subcultivations  from  the  flasks 
showing  evidence  of  growth,  to 
determine  that  no  contamin- 
ation has  taken  place. 
Spores. 

Moist  t.  d.  p.  is  that  time  ex- 
posure to  a  temperature  of  100 
C.  necessary  to  kill  all  spores 
present  in  a  suspension. 

Make  12  agar  slant  cultivations 
and  -  incubate  under  optimum 
conditions.  Examine  micro- 
scopically to  determine  the 
presence  of  spores.  Under 
sterile  conditions  place  about 
5  cc.  normal  saline  into  each 
tube  and  by  means  of  a  plat- 
inum wire  emulsify  the  surface 
g-rowth;  add  the  60  cc.  emulsion 
to  140  cc.  of  normal  saline  con- 
tained in  an  -  Erlenmeyer  flask; 
place  the  flask  in  a  water-bath 
of'  boiling  water.  When  the 
temperature  of  the  flask  reaches 
100°  C.  remove  by  means  of  a 
sterile  pipette  5  cc.  of  the  sus- 
pension from  which  plate  and 
flask  cultivations  are  made.  Re- 
peat process  intervals  of  25 
minutes  steaming.  Control  the 
experiments  by  removing  the 
suspension  at  intervals  of  or 
1  minute  during  the  5  or  10 
minutes  preceding  the  previous- 
ly determined  t.  d.  p. 

Dry  t.  d.  p.  Make  an  ag-ar  slant 
cultivation  and  incubate  under 
optimum  conditions  for  spore 
formation.  Under  sterile  con- 
ditions place  about  5  cc.  nor- 
mal saline  into  the  tube  and 
emulsify  the  growth.  Deter- 
mine the  presence  of  spores  by 
microscopic  examination.  Make 
12  cover-slip  films  and  place 
each  in  a  separate  Petri  dish. 


BACTERIOLOGY.  101 


Expose  each  dish  in  turn  for 
10  minutes  to  a  different  fixed 
temperature,  varying  5°  C.  be- 
tween 100°  C.  and  160°  C. 

Complete  examination  as  in  the 
vegetation  forms. 
.  REACTION  OF  MEDIUM. 

Baug-e.  Make  a  24  hour  bouillon 
culture  of  the  organism.  Pi- 
pette 0.1  cc.  of  the  cultivation 
into  a  tube  containing  9.9  cc. 
of  sterile  bouillon  and  mix. 
Inoculate  a  series  of  tubes  of 
nutrient  bouillon  of  varying 
reactions,  from  +  25  to  —  30, 
viz.:  +  25,  +  20,  +  15,  +  10. 
+  5,  neutral  —  5,  —  10,  —  15, 
—  20,  —  25,  —  30,  with  0.1  cc. 
of  the  diluted  cultivation  and 
incubat,e. 

Make  half  hour  observations 
from  the  third  to  the  twelfth 
hours  and  note  the  tube  or 
tubes  in  which  the  growth  first 
appears.  (Probably  the  opti- 
mum reaction).  At  the  end  of 
a  48  hour  incubation,  note  the 
extremes  of  acidity  and  akalin- 
ity  in  which  growth  has  taken 
place;  this  indicates  the  range 
^  of  reaction." 

Optimum  Reaction.  The  steps  are 
indicated  in  "range  reaction," 
but  must  be  fixed  within  nar- 
rower limits  by  inoculating  a 
series  of  tubes  which  have  a 
variation  in  reaction  of  1  in- 
stead of  5  for  five  points  on 
either  side  of  the  tube  or  tubes 
in  which  the  growth  first  ap- 
pears in  the  "range  reaction." 
RESISTANCE  TO  I^ETKA]^  AGENTS. 
Desiccation.  Make  an  agar-slant 
cultivation  and  incubate  for  48 
hours,  after  which  examine  to 
determine  the  absence  of  spores. 
Pipette  about  5  cc.  sterile  nor- 
mal saline  into  the  tube  and 
suspend  the  growth  in  it.  Make 
thin  spreads  of  the  suspension 
on  sterile  cover  slips  and  place 
them  inside  of  sterile  Petri- 
dishes  to  dry.  When  dry,  ele- 
vate the  lids  of  the  Petri-dish 
in  such  a  way  as  to  allow  ven- 
tilation and  place  the  dish  in  a 


BACTERIOLOGY. 


MuUer's  desiccator,  the  upper 
chamber  of  which  is  filled  with 
pure  H2  SO.,  cover  with  a  bell 
jar,  and  exhaust  the  air.  At  5 
hour  intervals  admit  air,  re- 
move a  cover  slip,  and  under 
sterile  conditions  tr^insfer  it  to 
a  culture  flask.  Reseal  the 
desiccator  and  exhaust  the  air. 
Incubate  the  culture  flasks  for 
7  days  if  necessary  and  pour 
plates  from  those  flasks  which 
show  growth  to  determine  the 
absence  of  contamination. 
Hourly  observations  should  now 
be  made  for  5  hours  preceding 
and  succeeding  the  death-time 
determined  above. 

iiigriit. 

(a)  Diffuse  Dayliffht.  Make  a  tube 
cultivation  in  nutrient  bouillon 
and  incubate  for  48  hours. 
Pour  10  gelatin  and  10  agar 
plates,  each  containing  0.1  cc. 
of  the  bouillon  culture.  Place 
one  agar  and  one  gelatin  plate 
into  the  hot  and  cold  incu- 
bators, respectively,  as  controls. 
On  the  center  of  the  cover  of 
the  remaining  plates  fasten  a 
black  paper,  cut  in  the  shape 
of  a  cross  or  some  other  figure, 
and  expose  the  plates  to  the 
action  of  diffuse  daylight,  for 
1,  2,  3,  4,  5,  6,  7,  8,  10,  12  hours, 
after  which  incubate  and  ex- 
amine after  24  and  48  hours 
and  compare  with  the  two  con- 
trols. If  the  growth  is  absent 
from  that  portion  of  the  plate 
not  covered  by  the  paper,  con- 
tinue the  incubation  and  daily 
observations  for  7  days. 

(b)  Direct  Stinligrlit.     Prepare  as 

above,  except  that  plates  ar^ 
placed  in  the  direct  rays  of  tht 
sun.  Stand  a  small  glass  disl 
(14  cm.  X  5  cm.)  on  top  of  eacl: 
plate  and  fill  with  a  2%  watery 
solution  of  potash  alum  (to  ab 
sorb  the  heat  of  sun's  rays) 
Make  exposure  and  incubate  a: 
in  experiment  above. 

(c)  Colours.     Test    separately  witl 

violet,  blue,  green,  red,  orang- 
and  yellow.    Prepare  plate  cul 


BACTERIOLOGY.  103 


tivations  as  in  "light"  experi- 
ments. Fasten  a  strip  of  black 
paper  (3  cm.  wide)  across  the 
cover  of  each  plate;  then  paint 
the  colors  over  the  remaining 
portions  of  the  cover.  The 
colors  are  prepared  by  placing 
2%  of  the  following  dyes: — 

Chrysoidine  (red),  aurantia  (or- 
ange), naples  yellow  (yellow), 
malachite  green  (green),  bluish 
eosin  (blue),  and  methylviolet 
(violet),  in  pure  photographic 
collodion. 

Expose  the  plates  to  bright  day- 
light for  varying  periods  as  in 
preceding  experiments. 

Heat.     (See  thermal  d.  p.) 

Antiseptic  and  Disinfectants.  Bi- 
chloride of  mercury,  formalde- 
hyde, carbolic  acid  are  general- 
ly selected  for  the  test,  noting 
the  strength  of  solution  and 
duration  of  exposure  necessary 
to  produce  death. 

They  are  examined  with  refer- 
ence to 

(a)  Inhibition  Coefficient, — that  %  of 
disinfectant  present  in  the  me- 
dium which  is  sufficient  to  pre- 
vent the  growth  and  multiplica- 
tion of  bacteria  therein. 
Prepare  a  series  of  6  bouillon 
cultivations  of  an  organism  in 
tubes  containing  10  cc.  of  me- 
dium.    Mark    the    tubes  from 

1  to  6;  place  in  No.  1  tube  — 

2  cc.  of  a  5%  carbolic  acid  so- 
lution (1:100);  in  No.  2  —  1  cc. 
(1:200);  in  No.  3  —  0.6  cc. 
(1:300);  in  No.  4  —  0.5  cc. 
(1:400);  in  No.  5  —  0.4  cc. 
(1-500);  and  in  No.  6  —  0.6  cc. 
(1:1000). 

Prepare  a  series  of  6  bouillon  cul- 
tiva^tions,  using 


Prepare  a  series  of  6  bouillon  cul- 
tivations, using 


104  BACTERIOLOGY. 


Incubate  the  three  sets  and  ex- 
amine them  from  day  to  day 
for  7  days  and  note  those  tubes, 
if  any,  in  which  growth  takes 
place. 

(b)  Inferior  letlial  coefficient  —  the 

time  exposure  necessary  to  kill 
vegetative  forms  suspended  in 
water  at  20°  to  25°  C.  in  which 
the  disinfectant  is  present  in 
concentration  insufficient  to 
cause  plasmolysis. 
Prepare  48  hour  agar  slant  culti- 
vations of  each  of  the  "test" 
organisms  and  examine  to  de- 
termine the  absence  of  spores. 
Prepare  solutions  of  different 
percentages  of  each  disinfect- 
ant and  make  a  series  of  hang- 
ing-drop preparations  from  the 
culture,  using  the  different  per- 
centage to  prepare  the  emulsion 
on  each  cover  slip.  Examine 
these  under  the  microscope  to 
determine  the  strongest  solu- 
tion which  does  plasmolyze  the 
organism.  Make  control  pre- 
parations of  these  two  solli- 
tions  and  determine  the  per- 
centage to  be  tested.  Transfer 
10  cc.  of  sterile  water  into  the 
agar  cultures  and  suspend  the 
growth  in  it,  after  which  it  is 
transferred  to  a  flask  contain- 
ing 90  cc.  of  sterile  water  and 
well  shaken.  10  cc.  of  this  dilu- 
tion is  placed  into  each  of  10 
sterile  test  tubes;  one  is  placed 
in  the  20°  C.  incubator  as  a 
control;  to  each  of  the  other 
tubes  a  sufficient  quantity  of 
a  concentrated  solution  of  the 
disinfectant  is  added  to  pro- 
duce the  percentage  previously 
determined.  Incubate  at  20°  C. 
Remove  the  control  tube  and 
one  of  the  other  tubes  contain- 
ing the  disinfectant  at  hourly 
intervals;  make  subcultivations 
upon  agar  and  incubate.  Ex- 
amine these  cultures  from  day 
to  day  for  7  days  and  determine 
the  shortest  exposure  necessary 
to  cause  the  death  of  vegeta- 
tive forms. 


BACTERIOLOGY^  105 

(c)  Superior  lethal  coefficient,  that 
time  exposure  necessary  to  kill 
spores  suspended  in  water  at 
20°  to  25°  C.  in  which  the  dis- 
infectant is  present  in  concen- 
tration insufficient  to  cause 
plasmolysis. 
Make  agar-slant  cultivations  of 
the  "test"  organism  and  incu- 
bate under  conditions  previous- 
ly determined  for  the  formation 
of  spores. 
Employ  that  percentage  solution 
of  the  disinfectant  determined 
in  the  inferior  lethal  coefficient 
and  complete  the  investigation 
as  detailed  therein,  increasing 
the  interval  between  planting 
the  subcultivations  to  two, 
three  or  five  hours  if  advisable. 
Where  it  is  necessary  to  leave 
the  organism  in  contact  with  a 
strong  solution  of  the  disin- 
fectant for  a  long  period  of 
time,  all  traces  of  the  disin- 
fectant must  be  removed  by 
several  centrif  ugations  and 
washing  of  the  bacteria  with 
sterile  water. 

5TUDY  OP  THE  FATKOGENICITY  OP 
AN  ORGANISM. 

•"or  the  study  of  the  pathogenicity,  the 
use  of  living  animals  has  become  a 
necessity  in  order  that  the — 

Pathogrenic  properties  of  bacteria 
already  isolated  in  pure  culture 
may  be  determined. 

The  conditions  influencing  the  vi- 
rulence of  an  organism  and  the 
pathogenic  effect  produced  by  its 
entrance  into  and  multiplication 
within  the  tissues  of  the  body, 
etc.,  can  only  be  carried  out  by 
animal  inoculation. 

Baising*  or  '^exalting"'  the  Virulence 
of  an  Organism. 

When  it  is  desired  to  raise  the  vi- 
rulence of  a  feebly  pathogenic 
organism,  one  of  the  following 
methods  of  inoculation  is  neces- 
sary— 

(a)  A  highly  susceptible  animal  is 
inoculated  with  a  pure  culture  of 
the    organism    and    then  passed 
,        from  animal  to  animal  as  rapidly 


106  BACTERIOLOGY. 

as  possible,  always  selecting  the 
method  of  inoculation  (e.  g. 
peritoneal)  which  places  the  or- 
ganism under  the  most  favorable 
conditions  for  growth  and  multi- 
plication. 

(b)  An  animal  is  inoculated  with  a 
pure  culture  of  the  organism  to- 
gether with  a  pure  culture  of 
some  other  organism,  which  is  in 
itself  of  a  virulency  sufficient  to 
produce  the  death  of  the  animal. 
The  inoculation  of  the  two  organ- 
isms may  be  made  into  the  same 
situation  or  in  different  parts  of 
the  body. 

By  the  association  of  these  two 
organisms,  the  one  of  low  viru- 
lence will  often  acquire  a  high 
virulence,  which  may  be  still 
further  raised  by  the  passage 
from  animal  to  animal. 

(c)  An  animal  is  inoculated  with  a 
pure  culture  of  an  organism  to- 
gether with  an  injection  of  a 
toxin  (e.  g.,  elaborated  by  the 
proteins  group,  etc.)  The  na- 
tural resistance  of 'the  animal  is 
lowered,  the  organism  allowed  to 
multiply,  its  virulence  is  then 
raised  by  passage  from  animal  to 
animal. 

Attenuating-  or  3^owering-  the  Viru- 
lence Qf  an  Orgranism. 

This  is  usually  brought  about  by 

(a)  Cultivating  the  organism  in  a 
media  unsuitable  by  reason  of  its 
composition  or  reaction. 

(b)  Cultivating  the  organism  in 
suitable  media  at  an  unsuitable 
temperature. 

(c)  Cultivating  the  organism  in 
suitable  media,  in  an  unsuitable 
atmosphere. 

(d)  Cultivating  the  organism  in 
suitable  media,  under  unfavorable 
conditions  as  to  light,  motion,  etc. 

(e)  Passing  the  organism  through 
naturally  resistant  animals. 

(f)  Exposing  the  organism  tc 
desiccation. 

(g)  Exposing  the  organism  to  gas- 
eous disinfections. 

(h)  By  a  combination  of  two  or 
more  of  the  above  methods. 


BACTERIOLOGY.  107 


II.  Isolation  of  Fathogrenlc  Bacteria. 

Certain  parasitic  bacteria  are  with 
great  difficulty  isolated  from  as- 
sociated saprophytic  bacteria  by 
the  ordinary  culture  methods  by 
reason  of  the  difficulty  with 
which  they  grow  upon  artificial 
culture  media. 

If  the  parasite  and  its  associated 
saprophyte  are  injected  into  an 
animal  susceptible  to  the  paras- 
ite, the  pathogenic  organism  can 
readily  be  Isolated  from  the  tis- 
sues of  the  infected  animal. 

Special  media  may  be  excellent  for 
the  growth  of  these  certain 
parasites,  but  the  associated 
saprophytes  will  also  grow  so 
abundantly  as  to  overgrow  the 
parasite;  therefore,  if  the  ma- 
terial containing  the  parasitic  or- 
ganism is  inoculated  under  the 
skin  of  a  susceptible  animal,  the 
pathogenic  organism,  in  a  few 
days,  will  have  entered  the  blood 
stream  and  killed  the  animal, 
leaving  the  saprophytes  at  the 
seat  of  inoculation. 

Cultivations  made  at  post  mortem 
from  the  animal's  heart  blood 
will  produce  a  pure  growth  of  the 
pathogenic  organism. 

In  obtaining  the  culture  from  the 
infected  animal,  complete  asepsis 
must  be  the  rule.  The  fur  or 
feathers  are  drenched  with  a  2% 
lysol  solution,  to  prevent  the 
hairs  from  flying  about  and  enter- 
ing the  body  cavities  during  the 
autopsy,  and  also  to  render  in- 
ocuous  any  vermin  that  may  be 
present.  With  sterile  forceps 
and  scalpel  incise  and  reflect  the 
skin.  Sear  the  whole  exposed 
surface  with  heated  searing  irons; 
remove  a  part  of  the  body  wall 
with  a  new  set  of  sterile  instru- 
ments and  proceed  to  make  the 
culture. 

III.  Identification  of  Pathog-enic  Bac- 

teria. 

The  morphological  and  cultural  re- 
semblances of  certain  pathogenic 
bacteria  are  in  some  cases  so 
great  as  to  make  identification 
impossible;    the    same  organism 


108  BACTERIOLOGY. 


may  under  carying-  condition  take 
on  an  appearance  so  different 
from  the  typical  that  results  are 
very  confusing".  A  simple  inocu- 
lation may  decide  the  point  at 
once. 

IV.    Study    of    the    Problems    of  Im- 
munity. 

It  is  only  by  careful  study  of  the 
behavior  of  the  animal  cell  and 
the  body  fluids  together  with  the 
infecting  organism  that  we  may 
understand  the  complex  problem 
whereby  the  cell  successfully  re- 
sists the  invading  organism. 
During  the  inoculation  studies, 
instances  of  both  racial  and  indi- 
vidual natural  immunity  will  be 
met  with.  Natural  immunity, 
however,  is  relative  only  and 
never  absolute  and  an  organism 
must  not  be  put  down  as  non- 
pathogenic until  many  different 
methods  of  inoculation  have  been 
accomplished  upon  animals  of 
different  species.     (See  immunity.) 

METHODS    USED    POR   THE  STUDY 
OP     THE     PATHOGENICITY  OP 
.   AIT  ORGANISM: 

1.    The  livings  Orgfanism. 

(a)  The     Psychrophilic  Bacteria. 

(Grow  only  at  or  below  18°  — 
20°  C.) 

Cultivations  are  prepared  under 
optimum  conditions  in  nutrient 
broth,  and  after  seven  days'  in- 
cubation, an  amount  of  the  cul- 
ture corresponding  to  1%  of  the 
body  weight  of  a  frog  is  injected 
into  the  frogs  dorsal  lymph  sac. 

Observe,  if  necessary,  for  28  days. 
(See  Animal  Inoculation). 

(b)  Mesophilic  Bacteria  (grow  at 
35°  —  37''  C.) 

Cultivations  are  prepared  under 
•  optimum  conditions  in  nutrient 
broth,  and  after  48  hours  incu- 
bation, inoculate  a  white  mouse 
subcutaneously  at  the  root  of  the 
tail, ,  with  an  amount  of  the  cul- 
ture corresponding  to  1%  of  its 
body  weight.  Inoculate  a  second 
mouse  of  the  same  weight,  or 
nearly  so,  ultraperitoneally  with 
a  similar  dose. 


BACTERIOLOGY.  109 


Observe  carefully  until  death  oc- 
curs, or  for  28  days.  (See  Animal 
Inoculation). 

If  death  takes  place  a  post-mortem 
examination  is  to  be  made.  If 
death  takes  place  shortly  after 
the  injection,  the  inoculation  ex- 
periment should  be  repeated  two 
or  three  times;  and  if  the  or- 
ganism invariably  exhibits  patho- 
genic effects,  ascertain,  if  possi- 
ble, its  minimal  lethal  dose  (from 
the  g-rowth  upon  solid  media)  for 
frogs  or  white  mice,  respectively. 
Now  make  tests  on  white  rats, 
guinea  pigs  and  rabbits. 
The  Toxins. 

Prepare  bulk  cultivations  of  the 
organism  in  glucose  *  formate 
broth  and  incubate  for  14  days 
under  optimum  conditions.  For 
the  determination  of: 

(a)  Intracellular  or  Insolul>le  Tox- 
ins. 

Heat  the  fluid  culture  in  a  water 
bath  at  60°  C.  for  30  minutes. 
Inoculate  a  tube  of  sterile  bouil- 
lon with  an  equal  amount  of  the 
heated  culture  and  incubate  under 
optimum  conditions  to  demon- 
strate the  absence  of  the  living 
organism. 

Inject  intravenuously  that  amount 
of  the  cultivation  corresponding 
to  1%  of  the  body  weight  of  the 
selected  animal. 

Observe  during  life,  or  until  the 
28  th  day,  and  in  the  event  of 
death  make  a  complete  post  mor- 
tem examination. 

The  experiment  should  be  repeated, 
and  if  a  positive  result  is  ob- 
tained, the  minimal  lethal  does 
of  "killed  culture"   is  estimated. 

(b)  Extracellular  or  Soluble  Toxins. 
Filter    the    cultivation    through  a 

porcelain  filter  into  a  sterile  filter 
f  flask.  Inoculate  the  various  an- 
imals subcutaneously  with  a 
quantity  corresponding  to  1%  of 
the  body  weight,  and  observe,  if 
necessary,  for  28  days. 
Inoculate  a  control  tube  of  bouillon 
and  incubate  to  demonstrate  the 
absence  of  living  organisms. 


110  BACTERIOLOGY. 


Repeat  the  experiment,  and  if  a 
positive  result  is  obtained  .de- 
termine the  minimal  lethal  dose 
of  the  toxin. 

ANIMAI.  INOCUI^ATION. 

Animals  employed  in  the  study  of 
pathogeneses  are  the  cold  blooded 
frogr;  toad  and  lizard;  the  warm 
blooded  mouse,  rat,  guinea  pig,  rabbit 
and  monkey;  the  hot  blooded  fowl 
and  pigeon. 

Before  animals  are  inoculated  they 
should  be  carefully  examined  so  as 
to  avoid  the  employment  of  any  al- 
ready diseased.  This  examination 
should  take  in  the  observation  of  the 
animal  at  rest  and  in  motion;  the  ap- 
pearance of  the  fur,  feathers  or 
scales,  inspection  of  the  eyes,  and  of 
the  external  orifices  of  the  body; 
tactile  examination  of  the  body  and 
limbs,  palpation  of  the  groins  and 
abdomen  and  in  many  cases  micro- 
scopical examination  of  fresh  and 
stained  blood-films. 

The  mouse  and  rat  may  suffer  from 
septicemia,  the  cysticercus  form  of 
taenia  murina.;  the  cystic  form  of 
.taenia  crassicollis  has  its  habitat  in 
their  lives:  scabies;  favus  and  try- 
panosoma  Lewisi. 

The  guinea  pig  may  suffer  from  scabies, 
coccidiosis  and  tuberculosis.  It  is 
well  to  test  the  animal  by  injecting 
0.5  cc.  of  Koch's  old  tuberculin,  which 
will  cause  death  in  those  diseased 
within  48  hours. 

The  rabbit  may  suffer  from  psoric  acari. 
One  form  (sarcoptes  minor)  first 
shows  itself  as  yellowish  scales  and 
crusts  around  the  nose,  mouth  and 
eyes,  spreads  to  the  bases  and  outer 
surfaces  of  the  ears,  to  the  fore  and 
hind  limbs  and  into  the  groins  and 
around  the  genitals.  Another  form 
(psoroptes  communis  cuniculi)  com- 
mences at  the  bottom  of  the  concha  in 
the  form  of  white,  yellowish  masses 
of  crusts,  scales,  feces  and  dead 
acari.  The  coccidium  oviforme  is  a 
frequent  infection.  Infection  with 
ordinary  pyogenic  bacteria  frequently 
occurs  in  the  rabbit. 
The  monkey  is  very  prone  to  tuber- 
culosis and  should  be  injected  with 
1  cc.  Old  tuberculin. 


BACTERIOLOGY.  Ill 


Anematode  (oesophagostoma  inflatum) 
resembling  the  anchylostomum,  par- 
asitic in  cattle,  Is  frequently  present 
in  the  tissues  of  the  monkey,  may 
bore  through  the  intestinal  wall  and 
produce  small  cysts  in  the  mesentery. 

The  pigeon  may  be  infected  by  haemos- 
poridia  and  pigeon  diphtheria. 
The  fowl  may  suffer  from  scabies  and 
ringworm,  fowl  cholera  or  fowl  sep- 
ticaemia. 

Animal  inoculation  is  purely  surgical 
operation,  therefore,  in  its  perform- 
ance strict  attention  must  be  paid  to 
asepsis  and  precautions  adopted  to 
guard  against  contamination  of  the 
material  to  be  introduced  into  the 
animal. 

The  Material  used  for  Inoculation  may 

be 

1.  Cultures  of  bacteria  grown — 

(a)  In  fluid  media;  a  definite  meas- 
ured quantity  injected  by  means 
of  a  syringe  or  (if  a  large  bulk 
is  to  be  introduced)  by  means  of 
a  gravity  apparatus  consisting  of 
a  funnel,  rubber  tubing  and  an 
injection  needle. 

(b)  On   solid   medi^.;    a   fluid  sus- 
pension is  made  by  washing  the 
culture  with  a  little  bouillon  or 
normal  saline,  and  then  injected  i 
as  above. 

2.  Metabolic  products  of  bacteria  (Tox- 

ins). Prepared  as  previously  de- 
scribed and  injected  as  described 
under  cultures  of  bacteria. 

3.  Pathological  products   (fluid  secre- 

tions  and   excretions,    solid  tis- 
sues) are  treated  as  fluid  cultiva- 
tions.    If   the   material   is  very 
thick  a  small  portion  of  bouillon 
or  normal  saline  solution  may  be 
used  to  dilute  it.     Solid  tissues 
are  rubbed  up  in  a  sterile  mortar 
with  a  small  portion  of  bouillon. 
The  Methods  of  Inoculation. 
Tne  animal  is  held  firmly  by  an  assist- 
ant or  secured  to  an  operating  table, 
liquid  soap  applied  to  the  area  select- 
ed for  inoculation  with  a  small  pad 
and   lathered   freely   by   the   aid  of 
warm  water;  shave  thoroughly;  wash 
with  1%  lysol  solution;  wash  off  lysol 
with   ether   and  allow   the   ether  to 


112  BACTERIOLOGY. 


evaporate;  then  inoculate  by  method 
selected  from  the  following: 

1.  Cutaneous  MetlLOd.   (No  anaesthetic). 

Make  numerous  short  parallel 
superficial  incisions  with  the 
point  of  a  sterile  scalpel  and 
when  the  oozing  has  ceased,  rub 
the  inoculum  into  the  scarifica- 
tions. Cover  the  area  with  a  pad 
of  sterile  gause  secured  by  ad- 
hesive or  collodion. 

2.  Subcutaneous  Method.     (No  anaes- 

thetic if  inoculum  is  solid  ethyl 
chloride  spray). 

If  the  inoculum  is  fluid  pinch  up  a 
fold  of  skin  between  finger  and 
thumb  and  inject  with  a  hypor- 
dermic  syringe. 

If  the  inoculum  is  solid,  raise  a 
fold  of  the  skin  in  a  pair  of 
forceps  and  make  a  small  inci- 
sion. By  means  of  a  probe  make 
a  small  pocket  in  the  subcu- 
taneous tissue  and  introduce  the 
tissue  inoculum  into  it.  Close 
the  wound  in  the  skin  with  a  clip 
(Michel's)  or  a  suture  and  cover 
the  area  as  in  cutaneous  method. 

3.  *   Intramuscular.     No   anaesthetic  if 

the  inoculum  is  fluid  but  if  solid 
use  A.  C.  E.  anaesthetic.  The 
method  is  practically  the  same  as 
in  the  subcutaneous,  except  that 
the  injection  is  made  deep  into 
the  muscle. 

4.  Intraperitoneal.      (No  anaesthetic). 

For  liquid  inoculum  the  method  is 
essentially  the  same  as  in  the 
subcutaneous,  except  that  the  en- 
tire thickness  of  the.  abdominal 
walls  is  pinched  up  into  a  tri- 
angular fold.  Ascertain  that  there 
are  no  coils  of  intestine  included 
by  slipping  the  peritoneal  sur- 
faces one  over  the  other. 
For  the  solid  inoculum,  an  A.  C.  E. 
anaesthetic  is  ,  used  and  the 
aponeuroses  between  the  recti 
muscle  are  divided  upon  a  direc- 
tor, the  peritoneum  likewise,  the 
inoculum  introduced:  the  peri- 
toneum closed  with  Lembert's 
sutures;  the  aponeuroses  and  skin 
incision  are  closed  together  with 
interrupted  suture. 


BACTERIOLOGY.  113 


Intracranial.    (A.  C.  E.  anaesthetic). 

(a)  Subdural.     By    the    use    of  a 

trephine  open  the  skull  in  the 
parietal  seg^ment  at  the  point  of 
intersection  of  the  medium  line 
and  a  line  joining  the  posterior 
canthi,  perforate  the  dura  and 
with  a  syringe  deposit  the  ma- 
terial immediately  below  this 
membrane  carefully  so  as  to  pro- 
duce no  injury. 

(b)  Intracerebral.  Same  as  in  in- 
tracranial except  that  the  needle 
is  pushed  into  the  substance  of 
one  or  the  other  cerebral  hemis- 
pheres. 

Intraocular.  (Cocaine  anaesthetic).* 
Two  needles  are  fitted  to  a 
syringe.  One  is  attached  to  the 
syringe  and  the  required  dose  of 
inoculum  is  taken  into  it;  the 
needle  is  then  removed.  The  other 
needle  is  used  to  pierce  the  cornea, 
allowing  the  aqueous  to  escape 
through  it,  then  without  removal 
the  syringe  is  attached  and  the 
inoculation  is  made  into  the  an- 
terior chamber. 

Intrapulmonary.  (No  anaesthetic). 
The  fluid  inoculum  is  injected 
through  the  5th  and  6th  inter- 
costal space  into  the  lung  tissue. 

Intravenous.  (No  anaesthetic).  The 
The  fluid  inoculum  must  be  pre- 
pared with  care  in  order  that 
when  injected  a  fatal  embolism 
may  be  obviated.  If  possible,  the 
fluid  should  be.  filtered  through 
sterile  filter  paper  to  do  away 
with  small  fragments  of  tissue. 
Eliminate  the  possibility  of  air 
bubbles.  After  the  usual  prepar- 
ation of  the  skin,  plunge  the 
needle  of  the  syringe  through  the 
skin  into  the  lumen  of  the  vein 
and  slowly  inject  the  inoculum. 
Withdraw  the  needle  and  press  a 
pledget  of  cotton  over  the  punc- 
ture. 

The  jugular  vein  may  be  utilized  in 
the  guinea  pig;  the  posterior 
auricular  vein  in  the  rabbit;  the 
internal  saphenous  vein  in  the 
dog  or  monkey. 

Inhalation  (No  anaesthetic).  The 
animal    is    placed    in    a  closed 


114  BACTERIOLOGY. 


metal  box  and  through  a  hole  in 
one  side  of  it  the  nozzle  of  the 
spraying  apparatus  (ordinary 
nasal  spray  will  do)  containing 
the  fluid  inoculum  is  introduced 
and  sprayed  into  the  interior  of 
the  box.  On  completion  of  the 
spraying,  the  animal  is  sprayed 
thoroughly  with  a  10%  solution 
of  formaldehyde  and  the  animal 
returned  to  its  cage.  The  inhala- 
tion chamber  is  thoroughly  disin- 
fected. In  another  method,  for 
both  fluid  and  powdered  inoculum, 
frequently  used,  a  wooden  gag 
provided  with  a  square  orifice 
through  which  a  tracheal  or 
oesophageal  tube  may  be  passed 
down  through  the  larynx  into  the 
trachea.  Connect  the  straight 
portion  of  a  Y-shaped  tube  to  the 
laryngeal  tube;  couple  one  branch 
of  this  to  a  separatory  funnel 
containing  the  fluid  inoculum  or 
insufflator  containing  the  powder- 
ed inoculum  and  the  other  to  a 
hand  bellows.  Allow  the  fluid 
inoculum  to  run  down  into  the 
lungs  by  gravity,  or  below  the 
powdered  inoculum  into  the  lungs 
by  means  of  a  bellows. 

10.  Intrag'astric.    (No  anaesthetic).  By 

use  of  a  gag  similar  to  the  one 
mentioned  above,  insert  a  soft 
rubber  catheter  into  the  stomach 
and  allow  a  measured  quantity  of 
the  inoculum  to  run  down  into  the 
stomach.  With  some  sterile  salt 
solution  wash  out  the  last  traces 
of  the  inoculum  in  the  catheter 
and  then  withdraw  it. 

11.  Feeding'.    Pieces  of  sterilized  bread 

are  soaked  in  the  fluid  inoculum, 
or  small  pieces  of  tissue  inoculum 
are  placed  in  sterile  dishes  and 
offered  to  the  animal. 
The  possession  of  pathogenic  prop- 
erties by  an  organism  is  indicated 
by  the  infection  of  the  experi- 
mental animal.  Infection  is  con- 
sidered to  have  taken  place 

(a)  When  the  death  of  the  animal 
is  produced  by  the  inoculum. 

(b)  When,  without  producing  death, 
the  inoculum  causes  local  or  gen- 


BACTERIOLOGY.  115 


eral  changes  of  a  pathological 
character, 
(c)  When  either  with  or  without 
death,  or  the  production  of  local 
or  general  changes,  certain  sub- 
stances make  their  appearance  in 
the  body  fluids  which  can  be 
shown  to  exert  some  specific  ef- 
fect when  brought  into  contact 
with  cultivations  of  the  organism 
originally  inoculated. 

The  observation  upon  the  animals 
inoculated  must  begin  immediate- 
ly and  only  terminate  with  the 
death  of  the  animal.  If  the  an- 
imal appears  to  be  unaffected  it 
should  be  killed  at  the  end  of 
2  or  3  months  and  a  complete 
post-mortem  carried  out. 

The  examination  of  the  animal 
should  consist  of 

(a)  General  Observation  daily  as  to 
general  appearance,  weight,  and 
temperature. 

(b)  Special  Observations. 

1.  As  weekly  examination  of  the 
site  of  inoculation  and  the  neigh- 
boring glands  palpated. 

2.  As  to  any  local  reaction  (sup- 
puration carefully  examined  both 
microscopically  and  culturally). 

3.  Frequent  examination  of  the 
blood  histologically. 

4.  Examination  of  the  blood  bac- 
teriologically  for  the  presence  of 
the  organism  previously  injected 
into  the  animal. 

Method: 

Sterilize  a  glass  syringe  and  moisten 
its  interior  with  a  sterile  solution 
of  sodium  citrate  (sodium  citrate 
10  gm.,  sodium  chloride  0.75  gm., 
distilled  water,  100  cc.)  If  more 
than  5  cc.  of  blood  is  required,  re- 
tain about  V2  cc.  of  the  sodium 
citrate  solution  in  the  syringe  to 
prevent  coagulation  of  the  blood. 
Prepare  the  animal  and  introduce 
the  syringe  needle  into  the  lumen 
of  the  selected  vein;  collect  suf- 
ficient blood;  withdraw  the  needle; 
deliver  the  citrated  blood  into  a 
flask  containing  250  cc.  of  nu- 
trient broth  and  incubate  until 
growth  occurs  or  until  the  expi- 
ration of  10  days. 


116  BACTERIOLOGY. 


5.  Examination  of  the  blood  sero- 
logfically  to  demonstrate  the  pres- 
ence of  antiV3odies  as  antitoxin, 
agg'lutinin,  precipitin,  opsonin, 
and  immune  body  or  bacteriolysin. 
(See  under  immunization). 
Conditions  Necessary  to  Infection  are 
1.  The  micro-org-anism  must  "be  patho- 
g*enic.  It  must  be  a  parasite. 
Organisms  that  are  parasitic  are 
not  necessarily  pathogenic;  how- 
ever, certain  requirements  must 
be  met  in  order  that  an  organism 
may  be  in/ectious  for  any  given 
animal,  and  by  this  is  meant,  the 
ability  of  an  organism  to  live  and 
multiply  in  the  animal  fluids  and 
tissues. 

Organisms  which  do  not  grow  at 
body  temperature  are  not  patho- 
genic, neither  are  the  strictly 
aerobic  organisms  as  they  are 
not  able  to  obtain  oxygen  in 
available  combination  from  carbo- 
hydrates. Aerobic  organisms  are 
practically  unable  to  multiply  in 
the  blood  stream  and  produce 
general  infection. 
2.,   The  org'anism  must  "be  virulent. 

Pathogenic  organisms  differ  very 
much  in  their  power  to  incite 
disease.  This  variation  in  viru- 
lence occurs  not  only  among  dif- 
ferent species  of  pathogenic  or- 
ganisms, but  may  occur  within 
the  same  species.  Certain  organ- 
isms when  kept  upon  artificial 
media  or  in  unfavorable  environ- 
ment for  some  time,  are  much 
less  virulent  than  those  isolated 
from  the  bodies  of  man  or  an- 
imal. 

3.  The    number    of    ors^anisms  which 

g'ain  entrance  to  the  animal  tissue 
must  be  of  sufficient  number. 

A  small  number  of  organisms,  even 
though  of  the  proper  species  and 
of  sufficient  virulence,  may  be 
overcome  by  the  defenses  of  the 
body.  The  more  virulent  the  or- 
ganism, the  smaller  the  number 
necessary  to  produce  disease. 

4.  An  Infection  Path  by  which  bacteria 

gain  entrance  is  of  importance  in 
determining  whether  or  not  dis- 
ease   will     occur.  Streptocpcci 


BACTERIOLOGY.  117 


when  swallowed  may  cause  no  ef- 
fect, while  if  rubbed  into  the 
abraded  skin  will  give  rise  to  a 
severe  reaction.  Typhoid  rubbed 
into  the  skin  may  not  give  rise 
to  any  reaction  of  moment,  while 
if  swallowed  may  .cause  fatal  in- 
fection. Animals  are  protected 
from  bacterial  invasion  by  the 
skin  and  mucous  membrane,  and 
when  these  are  healthy  and  unin- 
jured, micro-organisms  are  usual- 
ly kept  out,  though  they  may  oc- 
casionally pass  through  uninjured 
skin  and  mucosa.  There  can  not 
be  much  doubt  that  the  tubercle 
bacilli  may  pass  through  the  in- 
testinal mucosa  into  the  lymph- 
atics without  causing  local  lesion. 
5.    Animal  must  be  susceptilble. 

Susceptibility  is  relative  and  not 
absolute.  It  may  be  natural  to 
a  certain  race;  it  may  be  acquired 
by  the  presence  of  conditions 
which  lower  vitality;  it  may  be 
inherited,  by  reason  of  an  in- 
herited tendency. 
Even  though  virulent  pathogenic 
organisms  may  pass  through  an 
injured  portion  of  the  skin  or 
mucosa,  it  does  not  necessarily 
follow  that  infection  will  take 
place,  as  animals  have  an  im- 
munity (see  "Immunity")  which, 
if  normally  vigorous  and  active, 
will  usually  overcome  a  certain 
number  of  the  invading  organ- 
isms. If  this  immunity  is  weak 
by  reason  of  depression,  or  the 
invading  microorganisms  are  very 
virulent  or  plentiful,  infection 
takes  place. 


INFECTIONS. 

When  microorganisms  have  gained  an 
entrance  into  the  animal  body  and 
give  rise  to  disease,  the  process  is 
spoken  of  as  infection. 

In  contact  with  the  body  of  animals  is 
a  vast  flora  of  microorganisnis,  some 
constant  parasites,  some  transient  in- 
vaders, some  harmless  saprophytes 
and  some  capable  of  becoming  patho- 
genic. 


118  BACTERIOLOGY. 


The  phenomena  of  infection  are  reac- 
tions between  the  microorganism  and 
the  body  defense. 

In  order  to  cause  infection  bacteria 
must  gain  entrance  to  the  body  by 
paths  adapted  to  their  own  cultural 
requirements  and  must  be  permitted 
to  multiply. 

They  may  then  give  rise  merely  to 
local  inflammation,  necrosis  and  ab- 
scess formation;  they  may  remain  at 
the  point  of  entrance  and  elaborate 
toxins  which  are  absorbed  and  cir- 
culated by  the  blood;  they  may,  from 
tne  local  lesion,  gain  entrance  into 
the  lymphatics  and  blood  vessels  and 
be  carried  freely  into  the  circulation, 
where,  if  they  survive,  bacteriaemia 
or  septicaemia  follows;  they  may  be 
carried  by  the  blood  to  other  parts  of 
the  body  and  find  lodgment  in  any 
of  the  organs  and  give  rise  to  sec- 
ondary foci  of  inflammation,  necrosis, 
and  abscess  formation  (pyemia). 

The  disease  arising  as  the  result  of  the 
infection  may  depend  wholly  or  in 
part  upon  the  mechanical  injury  pro- 
duced  by   the   inflammatory  process, 

.  the  disturbed  function  caused  by  the 
presence  of  the  bacteria  in  capillaries 
and  tissues,  and  by  the  absorption  of 
the  products  resulting  from  the  reac- 
tion between  the  body  cells  and  the 
bacteria. 

The  symptoms  characteristic  of  in- 
fectious diseases,  to  a  large  extent, 
result  from  the  abosrption  of: — 

Bacterial  Poisons,  produced  by  the  or- 
ganisms themselves. 

(a)  Ptomaines     were    discovered  by 

Brieger  during  his  investigations 
into  the*  nature  of  the  poisons 
evident  in  bacterial  infections. 
These  bodies  isolated  by  him 
from  decomposing  beef,  fish  and 
human  cadavers,  although  pro- 
duced from  proteid  material  by 
bacterial  action,  and  the  cleavage 
products  derived  from  the  culture 
medium,  they  are  not  true  bac- 
terial poisons  in  the  sense  in 
which  the  term  is  now  employed. 

(b)  Toxins.     The  poisons  produced  by 

all  pathogenic  microorganisms  are 
soluble,  secretory  products  of  the 
bacterial   cell,   passing   from  the 


BACTERIOLOGY.  119 


cell  into  the  culture  medium  dur- 
ing their  life. 

They  may  be  obtained  free  from  the 
bacteria  by  filtration  and  in  a 
purer  state  from  the  filtrate  by 
chemical  precipitation,  etc. 

The  bacillus  of  diphtheria  and  the 
bacillus  of  tetanus  are  examples. 
If  a  several  day  bouillon  growth 
of  these  organisms  is  passed 
through  porcelain  filters,  the  fil- 
trate will  often  be  extremely 
toxic,  while  the  residue  will  be 
inactive  or  very  weak.  If  the 
residue  possesses  any  toxicity  at 
all,  the  symptoms  appearing  will 
be  quite  different  from  those  pro- 
duced by  the  filtrate.  Other  or- 
ganisms act  in  an  opposed  man- 
ner, e.  g.,  spirillum  cholera  and 
bacfillus  typhosus.  If  these  are 
cultivated  and  filtered,  the  filtrate 
will  be  toxic  only  to  a  slight  de- 
gree, while  their  residue  may  be 
very  toxic.  This  is  evidently  due 
to  poisons  not  secreted  into  the 
medium  but  rather  attached  to 
the  bacterial  body.  They  are 
termed  endotoxins,  and  the 
greater  number  of  pathogenic 
bacteria  seem  to  act  under  this 
class. 

Mode  of  action  of  bacterial  poisons 

is  much  the  same  as  the  ability 
of  the  various  narcotics  and  al- 
kaloids to  select  special  tissues 
or  organs  and  enter  into  a  com- 
bination with  them,  either  chem- 
ically or  physically,  or  both. 

Soluble  toxins  like  the  bacillus  of 
tetanus  and  the  botulinus  bacil- 
lus attack  specifically  the  nervous 
system.  Certain  poisons  elabor- 
ated by  certain  organisms  as  the 
staphylococci,  streptococci,  etc., 
attack  the  red  blood  cell  (haemo- 
lysin)  while  others  attack  the 
white  blood  cell  (leukocidin"). 

These  toxins  when  in  solution  can 
be  removed  by  the  acjdition  of 
their  specific  tissue,  e.  g.,  solution 
of  tetanus  toxin,  if  treated  with 
brain  substance  and  centrifuged 
leaves  the  solution  free  from 
toxin;  likewise  haemoly tic  poisons 
can  be  removed  from  solutions  by 


120  BACTERIOLOGY. 


contact  with  red  blood  cells,  but 
only  when  the  red  blood  cells  of 
a  susceptible  species  are  em- 
ployed. 


THEORY  OF  IMMUNITY. 

Several  theories  have  been  advanced  to 
account  for  the  various  phenomena 
of  immunity.     Pasteur  advanced  the 

"Ezliaustion  theory,"  in  which  bacteria 
by  their  growth  in  the  body  used  up 
or  exhausted  something  vitally  neces- 
sary to  their  subsequent  growth. 

detention  theory  in  which  certain 
noxious  agents  are  retained  by  the 
body,  which  prevent  further  growth 
of  bacteria. 

Cellular  or  hiologric  theory  of  Metchni- 
"koSf  or  "Phagocytosis." 

Humoral  or  chemical  theory  of  ZShrlich, 
or  "Side  Chain." 

The  theory  accepted  by  most  bacteri- 
ologists   is    a    combination    of  the 
theories  of  Metchnikoff  and  Ehrlich, 
and  is  called  the  Cellulo -humoral. 
The  theories  of  immunity,  accept- 
able   at    the    present    time,  are 
based    upon    two    branches  of 
.  study: — 

(1)  A  conception  formulated  by  the 
German  school  under  the  leader- 
ship of  Ehrlich,  Pfeiffer,  Kruse; 
deal  entirely  with  the  phenomena 
occurring  in  reaction  between  bac- 
teria or  bacterial  products  and 
body  fluids. 

(2)  The  participation  of  the  cellular 
elements  of  the  body  in  its  re- 
sistance to  infectious  organisms. 
Phagocytosis  was  formulated  by 
studies  at  the  Pasteur  Institute 
in  Paris,  under  the  leadership  of 
Metchnikoff. 


IMMUNITY. 

It  is  plain  that  the  mere  entrance  of 
pathogenic  organism  into  the  animal 
body  through  the  skin  or  mucosa  does 
not  necessarily  lead  to  the  develop- 
ment of  an  infection.  The  body  must 
therefore  possess  certain  means  of 
defense  in  order  that  the  pathogenic 
grerms  after  they  have  gained  en- 
trance into  the  tissue  and  fluids  will 


BACTERIOLOGY. 


121 


be  disposed  of,  or,  at  least,  be  pre- 
vented proliferating  and  elaborating 
their  poisons.  The  condition  which 
enables  the  body  to  accomplish  this 
is  spoken  of  as  resistance,  and  when 
this  resistance  is  especially  marked, 
it  is  spoken  of  as  ''immunity." 

Immunity,  therefore,  denotes  that  con- 
dition of  an  organism  which  enables 
it  to  resist  an  attack  of  the  particular 
bacteria  and  their  toxic  secretion 
against  which  they  are  said  to  be 
immune.  The  varieties  of  immunity 
are: — 

1.  Natural  Immunity,  as  an  inheritance 

from  immune  ancestors. 

(a)  Species  Immunity. — Many  infec- 
tious diseases  common  to  man 
do  not  occur  in  animals;  e.  g. 
gonorrhea  and  syphilis  do  not  oc- 
cur in  animals  except  when  pro- 
duced experimentally  and  this 
with  the  greatest  difficulty.  Lep- 
rosy, influenza,  etc.,  have  not  been 
transmitted  to  animals,  likewise 
human  beings  are  immune  to  dis- 
eases which  attack  animals. 

(b)  Racial  Immunity.  Separate  races, 
or  varieties  within  the  same  spe- 
cies, often  display  differences  in 
their  immunities  towards  patho- ' 
genie  organisms;  e.  g.,  Algerian 
sheep  show  a  much  higher  resist- 
ance to  anthrax  than  do  our 
domestic  sheep.  The  difference  in 
resistance  towards  tuberculosis 
between  the  Caucasian  and  the 
American  Indian,  the  Eskimo  and 
Negro  is  very  striking.  Converse- 
ly, the  comparative  immunity  of 
the  negro  from  yellow  fever, 
which  shows  very  great  virulence 
toward  the  Caucasian. 

(c)  Individual  Immunity  Is  noticed  to 
some  extent  in  man  and  may 
probably  be  attributed  to  indi- 
vidual variation  in  the  body 
metabolism;  e.  g.,  depressions  in 
gastric  acidity  predispose  to  in- 
fection of  gastrointestinal  origin; 
anatomical  differences  may  act  as 
predisposing  factors  towards  in- 
fection. 

2.  Acquired  Immunity. 

(a)  Active  Immunity  is^naturally  ac- 
quired by  having  had  a  previous 


122  BACTERIOLOGY. 


infection.  This  is  illustrated  by 
an  infection  with  typhoid  fever, 
yellow  fever,  and  many  of  the 
exanthemata.  A  single  attack  ot 
any  of  these  diseases  protects 
the  individual  for  a  limited  period 
and  frequently  for  life. 
It  may  be  artificially  acquired  by: 

(1)  Inoculations  with  weakened,  at- 
tenuated cultures  of  bacteria. 

(2)  Inoculation  with  sublethal  doses 
of  fully  virulent  microorganisms. 
Successive  inoculations  with 
gradually  increased  doses  of  the 
virus  creates  an  immunity  suffi- 
cient to  resist  ten  times  the  toxic 
dose. 

(3)  Injecting  with  gradually  in- 
creased doses  of  dead  microorgan- 
ism. Used  especially  in  that 
class  of  bacteria  in  which  the 
cell  bodies  (endo  toxins)  had  been 
found  to  be  more  poisonous  than 
their  extra-cellular  products 
(toxins). 

(4)  Injecting  gradually  increased 
doses  of  the  bacterial  product 
(toxins). 

(b)  Passive  Imnmnity  is  acquired  by 
injection  of  the  serum  of  animals 
that  have  been  rendered  immune 
by  artificial  methods,  into  the  in- 
dividual infected  or  to  be  pro- 
tected. 

This  type  of  immunity  is  used 
chiefly  against  diseases  caused  by 
bacteria  which  produce  powerful 
toxins,  and  the  sera  of  animals 
immunized  against  such  toxins 
are  called  antitoxic  sera. 

Passive  immunity  against  micro- 
organisms that  do  not  have  mark- 
ed toxin  formation  has  not  been 
successful.  The  microorganisms 
which  are  injurious  by  reason  oi 
the  content  of  the  bacteria  cell, 
rather  than  by  the  secreted 
soluble  toxins,  probably  do  not 
produce  antitoxins  in  the  sera  oi 
immunized  animals.  . 

The  substances  produced  by  their 
immunization  seem  directed 
against  the  invading  organisms  in 
that  they  have  the  power  of  de- 
stroying the  specific  germ  used  m 
the  production  of  immunity. 


BACTERIOLOGY.  123 


The  anti  bacterial  sera  are  used  in 
laboratory  animals  to  immunize 
them  against  a  large  number  of 
^^erms,  and  if  used  just  before, 
at  the  same  time  or  soon  after 
infection,  they  seem  fairly  ef- 
fective. 

In  human  disease  their  use  has  been 
disappointing,  except  when  the 
anti  bacterial  sera  could  be 
brought  in  direct  contact  with  the 
germs,  as  in  closed  cavities  or 
localized  lesions;  e.  g.,  Plexner's 
sera  for  meningitis. 


ANTIBODIES. 

The  treatment  of  the  animal  body  with 
bacteria  or  their  products  gives  rise 
to  a  variety  of  reactions  which  result 
in  the  presence  of  "antibodies."  These 
bodies  are  not  produced  by  bacteria 
or  their  products  only.  They  may  be 
produced  by  a  variety  of  poisons  of 
plant  and  animal  origin. 

Nuttall,  Fodor  and  Pluggs  (1886),  noted 
the  bacterial  properties  of  normal 
blood.  A  study  of  the  blood  sera  of 
immunized  animals  by  Beljarff  showed 
no  change  from  normal  as  to  index 
of  refraction,  specific  gravity,  and  al- 
kalinity. 

Joachim,  Moll,  Hiss  and  Atkinson  found 
immunized  blood  sera  richer  in  glob- 
ulin than  normal  serum. 

Very  little  light  was  thrown  upon  the 
phenomenon  of  immunity  until  Nut- 
tall,  Fodor  and  Buchner  demonstrated 
the  power  of  normal  blood  serum  to 
destroy  bacteria.  This  property  of 
the  blood  diminished  with  age  and 
was  destroyed  by  heating  to  56°  C. 
Buchner  called  this  themolabile  sub- 
stance alexin. 

Behring,  Kitasato  and  Wernicke,  in  1890 
and  1892,  showed  that  the  blood  sera 
of  actively  immunized  animals  against 
the  toxins  of  diphtheria  and  tetanus 
would  protect  normal  animals  against 
the  poisons  of  these  diseases.  Behring 
called  this  substance,  contained  in  the 
blood  sera  of  immunized  animals, 
antitoxins. 

Soon  after  this  Ehrlich  produced  anti- 
toxin against  some  of  the  higher 
plants.     Calmette  produced  antitoxin 


124  BACTERIOLOGY. 


against  snake  and  scorpion  poisons; 
Kempner  against  the  poison  of  bacil- 
lus botulinus,  etc. 

The  formation  of  antitoxins  directed 
against  the  soluble  toxins  did  not  ex- 
plain the  immunity  acquired  against 
bacteria  which  produced  no  soluble 
toxin.  Pfeifeer  (1894),  threw  light 
upon  this  when  he  injected  into  the 
peritoneal  cavity  of  cholera-immune 
guinea-pigs,  cholera  spirilla. 

The  microorganism  often  underwent 
complete  solution,  determined  by 
hanging-drop  preparations. 

MetchnikofC  and  Bordet  showed  that 
this  lytic  process  would  also  take 
place  in  vitro.  The  constituents  of 
the  blood  serum  which  gave  rise  to 
this  destructive  phenomenon  were 
called  'bacteriolysins. 

Gruber  and  Durham  then  discovered 
another  specific  property  of  immune 
serum  to  which  the  name  agrgrlutiniu 
was  applied.  Certain  bacteria,  when 
brought  into  contact  with  the  serum 
of  animals  immunized  against  them, 
became  clumped,  lost  their  motility 
and  firmly  agglutinated. 

In  1897,  Kraus  demonstrated  that  pre- 
cipitates were  formed  when  the  fil- 
trates of  cultures  of  typhoid,  cholera, 
etc.,  ^ere  mixed  with  their  specific 
immune  sera.  These  substances  he 
called  precipitins. 

The  large  variety  of  substances,  some 
poisonous,  some  inocuous,  that  pos- 
sess the  power  of  stimulating  anti- 
body formation  in  the  sera  of  animals 
are  termed  autigrens  or  antibody- 
producers. 

Ehrlich  proposes  three  forms  of  recep- 
tors in  explanation  of  all  varieties  of 
antibodies.     (See  Side  Chain  Theory). 
1st    order    liaptines    or  receptors, 
when  free  in  the  circulation,  con- 
stitute  the   antitoxins   and  anti- 
ferments. 
2nd    order   haptines    or  receptors, 
when  free  in  the  circulation,  serve 
as  anchorage  and  for  the  further 
digestion  of  antigens.     They  are 
precipitins  and  agglutinins. 
3rd    order    haptines    or  receptors 
merely  anchor  suitable  substances 
and  exert  no  action  till  combined 
with  the  complement.    When  free 


BACTERIOLOGY.  125 


in  the  circulation  with  a  chemical 
group  having"  affinity  for  the 
antig-en  and  a  complementophile 
group  are  the  amboceptors  of 
bacteriolytic,  cytolytic  and  hemo- 
lytic sera. 

The  second  group  of  receptors  which 
give  rise  to  agglutinins  and  pre- 
cipitins Ehrlich  believes  to  be  made 
up  of  a  single  hatophore  group  for 
the  anchorage  of  the  ingested  ma- 
terial, and  an  attached  zymophore 
group,  or  ferment,  which  changes  the 
anchored  substance  preparatory  to  its 
absorption  by  the  cell  protoplasm. 

Bordet  has  shown  that  it  is  not  the 
agglutining  itself  which  agglutinates, 
but  the  agglutinin  in  combination 
with  its  antigen  is  agglutinated  by 
the  salt  solution.  He  therefore  dis- 
agrees with  Ehrlich  and  concludes 
that  the  phenomenon  of  agglutination 
consists  of  the  union  of  the  antibody 
with  its  antigen  in  a  celloidal  solu- 
tion, and  that  the  actual  agglutina- 
tion is  a  secondary  phenomenon  de- 
pending possibly  upon  a  change  in  the 
physical  properties  of  the  emulsion. 
The  feame  he  holds  to  be  true  of  pre- 
cipitins. 

The  antibodies  which  can  be  demon- 
strated are  agglutinin,  precipitin,  op- 
sonin and  bacteriolysin. 

These  substances  cannot»be  isolated  in 
purity  apart  from  the  blood  serum, 
consequently,  methods  have  been 
elaborated  to  permit  of  their  recog- 
nition. 

The  serum  from  the  experimental  an- 
imal (specific  serum)  is  studied  and 
compared  with  the  serum  from  an 
uninoculated  animal  of  the  same  spe- 
cies (normal  serum).  In  order  that 
the  differences  existing  in  the  serum 
of  various  individuals  may  be  elim- 
inated, a  mixture  of  sera  obtained 
from  several  normal  animals  (pooled 
serum)  is  usually  used. 

Collection  of  Serum. 

Shave  the  dorsal  surface  of  the  ear, 
wash  with  lysol,  remove  lysol  by 
dropping  ether  over  it  and  allowing 
the  ether  to  evaporate,  puncture  the 
vein  and  collect  the  blood  by  means 
of  a  small  blood-collecting  pipette, 
touch  the  issuing  drop  of  blood  with 


126  BACTERIOLOGY. 


one  end  of  the  pipette,  which  is  held 
at  an  angle  so  that  the  blood  will 
flow  down  into  it.  When  the  tube  is 
about  two  thirds  full,  hold  it  by  the 
end  containing  the  blood,  the  clean 
end  pointing  obliquely  upward,  warm 
this  end  with  the  bunsen  flame  to 
expel  some  of  the  contained  air;  then 
seal  it  in  the  flame.  Shake  the  blood 
into  the  closed  end  and  seal  the  other 
end  in  the  flame. 

When  the  blood  has  clotted  place  the 
pipette  in  the  centrifuze,  the  end  first 
closed  pointing  downward,  and  cen- 
trifugalize  thoroughly.  The  blood 
cells  will  then  be  found  collected  in 
a  firm  mass  at  one  end,  and  above 
them  wil  appear  the  clear  serum. 

Mark  the  pipette  above  the  serum  with 
a  file  and  break  it  off  at  this  point; 
the  serum  is  now  accesible  for  test- 
ing. 


IMMUNIZATION. 

In  order  to  study  the  pathogenic  pow- 
ers of  any  particular  bacterium,  the 
active  immuniation  of  one  or  more 
normal  animals  becomes  necessary. 
This  is  done  by  various  methods; 
seldom  by  one  method  only,  but  usual- 
ly by  a  combination  of  methods 
adapted  to  suit  each  particular  case. 
The  ordinary  methods  used  are  as 
follows: 

1.  Active  Immunity.  (See  "Immunity"). 
An  illustration  of  how  the  general 
methods  of  immunization  are  carried 
out  is  as  follows: — 

A  full  grown  rabbit  weighing  from  1200 
to  1500  gms.  or  over  is  most  suitable 
for  immunization. 

A  small  rabbit  is  inoculated  intraperi- 
toneally  with  one  or  two  loopfuls  of 
2i  hour  optimum  culture  of  the  viru- 
lent organism  selected.  Death  should 
follow  within  24  hours,  or  at  most, 
in  48  hours.  Under  aseptic  precau- 
tions the  rabbit  is  "posted,"  and  a 
loopful  of  heart  blood  is  transferred 
to  50  cc.  of  sterile  broth.  This  is 
incubated  at  37°  C.  for  24  hours.  Also 
prepare  several  cultures  on  optimum 
media  from  the  heart  blood  of  the 
rabbit;  label  them  all  O.  C.  (original 
culture).     Incubate  at  37*  C.  for  24 


BACTERIOLOGY.  127 


hours,  after  which  seal  the  mouth  of 
the  plug-  tube  of  all  but  one  culture 
with  an  Indian  rubber  cap  painted 
with  shellac  or  paraffin,  and  replace 
in  incubator.  (Prevents  evaporation 
and  culture  will  remain  virulent  for 
a  considerable  period  of  time). 
Suspend  the  24  hour  broth  culture  in 
a  water  bath  at  60°  C.  for  one  hour 
in  order  to  kill  the  culture.  Cool 
immediately.  Now  determine  the  ster- 
ility of  the  cultivation  by  transferr- 
ing 1  cc.  to  each  of  several  tubes  of 
broth;  incubate  at  37°  C.  for  24  hours. 
If  a  growth  occurs,  heat  the  culture 
in  the  water  bath  again  at  60°  C.  for 

1  hour.  Test  again  for  sterility.  If 
sterile,  inject  the  suitable  animal 
mentioned   above   intravenously  with 

2  cc.  of  the  killed  culture;  also  inject 
10  cc.  into  the  peritoneal  cavity. 
Watch  the  animal  the  next  few  days; 
it  will  lose  some  weight  and  may 
show  pyrexia. 

When  the  temperature  and  weight  have 
become  normal  again,  inject  a  killed 
culture  in  a  mount  of  5  cc.  intraven- 
ously and  20  cc.  intraperitoneally. 
Weight  and  pyrexia  reaction  like  but 
less  marked  than  that  following  the 
first  inoculation  will  probably  follow. 

Subcultivate  on  optimum  medium,  the 
uncapped  O.  C. ;  incubate  for  24  hours 
at  37°  C. ;  determine  the  minimal 
lethal  dose  upon  a  number  of  mice. 

One  week  after  the  last  killed  culture 
injection,  prepare  a  fresh  optimum 
subculture  from  another  O.  C.  tube 
and  incubate  for  24  hours.  Prepare 
the  minimal  lethal  dose  and  inject 
subcutaneously  into  the  abdominal 
wall.  A  local  reaction,  a  pyrexia  and 
loss  in  weight  will  probably  be  ob- 
served. In  about  ten  days,  inject  a 
similar  minimal  lethal  dose  into  the 
peritoneal  cavity. 

Note  weight  and  temperature  of  animal 
carefully,  regulating  the  time  of  the 
animal's  inoculation  by  its  general 
condition  and  continue  to  inject  liv- 
ing cultivations  into  the  peritoneal 
cavity  in  gradually  increased  doses  by 
multiple  of  ten. 

At  intervals  of  2  months  the  animal's 
serum  is  tested  for  its  specific  anti- 
bodies. 


128  BACTERIOLOGY. 

Under  favorable  conditions,  after  6 
months'  work,  the  animal  may  be  in- 
jected intra  peritoneally  with  an 
entire  optimum  cultivation  of  the 
organism  without  any  ill  effect. 

The  animal  serum,  if  withdrawn  in 
about  a  week  following  the  injection, 
will,  if  injected  in  doses  of  0.01  cc. 
into  a  mouse,  protect  it  against  ten 
times  the  minimal  lethal  dose  of  the 
organism. 

Immunity  has  been  created  by  reason 
of  the  formation  of  an  antibody  spe- 
cific to  the  bacterium  in  question  and 
was  sufficient  in  amount  to  destroy 
enormous  doses  of  the  living  organ- 
ism, the  antigen  in  this  case  being 
a  bacterial  protoplasm  of  the  organ- 
ism with  its  endotoxin. 

If  death  did  not  immediately  follow  the 
injection  of  the  antigen,  specific  anti- 
bodies are  always  formed  in  greater 
or  lesser  extent;  and  in  experimental 
work  a  sufficient  amount  of  any  re- 
quired antibody  may  be  obtained 
without  carrying  the  process  of  im- 
munization to  completion.  * 

If  the  immunization  of  a  rabbit  toward 
bacillus  typhosus  be  carried  out  along 
the  lines  indicated  above,  it  will  be 
noticed  that  after  a  few  injections  of 
the  killed  cultivations  that  the  blood 
serum  of  the  animal  contains  specific 
agglutins  for   the  bacillus  typhosus, 

,and  if  the  object  of  the  experiment  has 
been  directed  toward  the  preparation 
of  agglutinin,  the  inoculation  may  be 
stopped,  even  though  the  animal  is 
not  yet  strictly  immune. 

Antibodies  may  also  be  formed  in  re- 
sponse to  antigens  other  than  micro- 
organisms, as  can  be  demonstrated 
by  the  injection  into  animals  of  for- 
eign proteins,  such  as  egg  albumin, 
blood  sera  or  the  red  blood  cells  from 
an  animal  of  different  species.  This 
will  lead  to  the  formation  of  specific 
antibodies  possessing  affinities  for 
their  specific  antigen.  Hemolysin  is 
a  common  antibody  of  this  type  and 
is  found  in  the  blood  serum  of  an 
animal  that  has  previously  been  in- 
jected with  the  washed  red  blood  cells 
from  an  animal  of  a  different  species. 
This  serum  will  possess  the  power 
of  disintegrating  the  blood  cells  of 


BACTERIOLOGY.  129 

the  variety  employed  as  antigen,  and 
cause  these  red  blood  cells  of  any 
other  species  of  animal. 
The  action  of  this  serum  is  due  to  the 
presence  of  two  distinct  bodies;  the 
one,  hemolysin,  and  the  other,  com- 
plement. 

Hemolysin  (immune  body,  copula,  sen- 
sitizingr  "body,  and  amboceptor)  is  a 

thermostable  antibody  which  is  form- 
ed by  the  repeated  injection  of  foreign 
red  blood  cells  into  an  animal.  Al- 
though it  is  itself  inert,  it  will  link 
up  the  complement  present  in  normal 
serum  to  the  red  blood  cells  of  the 
variety  used  as  antigen.  A  combina- 
tion of  the  two  results  is  Hemolysis. 

Hemolysin  is  obtained  by  collecting 
fresh  blood  serum  from  an  animal 
that  has  been  inoculated  with  ma- 
terial in  question  and  then  exposed 
to  a  temperature  of  56°  C.  for  15  to 
30  minutes  to  destroy  the  comple- 
ment. It  is  now  spoken  of  as  in- 
activated serum.  It  is  reactivated  by 
the  addition  of  fresh  normal  serum 
which  contains  the  complement. 

Although  hemolysin  is  of  importance  in 
making  clear  many  problems  of  im- 
munity, its  main  and  practical  im- 
portance is  in  its  application  of  the 
hemolytic  system  to  certain  laboratory 
methods  having  for  their  object  the 
identification  of  infected  entity  or  the 
diagnosis  of  existing  infection. 

For  its  use  directed  towards  the  meth- 
ods of  laboratory  diagnosis,  it  is  very 
convenient  to  prepare  hemolytic  serum 
specific  for  human  blood.  Ox  blood, 
sheep  blood,  or  goat  blood  may,  how- 
ever, be  used  instead. 

Complement  (or  alexin)  is  a  thermo- 
labile  oxidizable  body  present  in  the 
normal  serum  of  every  animal  in  a 
variable  but  unalterable  amount.  It 
is  a  substance  which  exerts  a  lytic 
effect  upon  all  foreign  matter  intro- 
duced into  the  blood  or  tissues.  It 
is,  in  itself,  comparatively  inert  and 
is  capable  of  exerting  its  greatest 
lytic  effect  only  when  in  the  presence 
of  and  in  combination  with  a  specific 
antibody  or  immune  body. 

It  is  obtained  by  collecting  fresh  blood 
serum  from  any  healthy  normal  an- 
imal. Guinea  pig  serum  is  most  fre- 
quently employed. 


130  BACTERIOLOGY. 


Preparation  of  hemolytic  sernm. 

Take  2  cc.  of  citrated  human  blood  col- 
lected from  a  vein  aseptically,  place 
it  in  the  centrifuge  and  centrifugalize 
it  thoroughly.  Wash  it  with  normal 
saline  and  centrifugalize  again.  Re- 
peat this  procedure  twice,  after  which, 
by  means  of  a  sterile  pipette,  transfer 
the  washed  blood  cells  into  a  sterile 
capsule.  Add  5  cc.  of  normal  saline 
and  mix  thoroughly.  By  means  of  a 
sterile  glass  syringe,  inject  the  blood 
suspension  into  the  peritoneal  cavity 
of  a  healthy  rabbit  weighing  at  least 
2500  gms.  At  the  end  of  seven  days, 
inject  the  washed  blood  cells  from 
10  cc.  of  human  blood  mixed  with 
5  cc.  of  normal  saline.  After  another 
interval  of  7  days,  repeat  the  injection 
of  washed  blood  cells  from  10  cc.  of 
human  blood  mixed  with  5  cc.  of 
normal  saline. 

After  5  days,  collect  about  2  cc.  of  the 
rabbit's  blood,  allow  it  to  clot,  sepa- 
rate the  serum  and  transfer  it  to  a 
sterile  test  tube.  Place  the  test  tube 
in  a  water  bath  of  56''  C.  for  15 
minutes  to  inactivate  the  serum,  then 
the  serum  quantitively  for  hemolytic 

.   properties  as  follows: — 

Titration  of  Hemolytic  Serum. 

1.  Two   test   tubes   marked  A   and  B 

each  containing  9  cc.  of  normal 
saline. 

2.  Add  1  cc.  of  rabbit  serum  to  tube 

A;  mix  thorouhgly. 
1  cc.  of  this  mixture  is  added  to 
tube  B;  mix  thoroughly. 

3.  Place    ten    small    test    tubes    in  a 

rack  and  number  them  from  1 
to  10. 

4.  By  means  of  a  pipette  place  into 
Tube  No.  1.     0.5    cc.   of  hemolytic 

serum  =  0.5  cc.  hemolytic  serum. 

Tube  No.  2.  0.2  cc.  of  hemolytic 
serum  =  0.1  cc.  hemolytic  serum. 

Tube  No.  3.  0.5  cc.  of  tube  A  mix- 
ture =1  0.05  cc.  hemolytic  serum. 

Tube  No.  4.  0.3  cc.  of  tube  A  mix- 
ture =  0.03  cc.  hemolytic  serum. 

Tube  No.  5.  0.2  cc.  of  tube  A  mix- 
ture =  0.02  cc.  hemolytic  serum. 

Tube  No.  6.  0.1  cc.  of  tube  A  mix- 
ture =  0.01  cc.  hemolytic  serum. 

Tube  No.  7.  0.5  cc.  of  tube  B  mix- 
ture =  0.005  cc.  hemolytic  serum. 


BACTERIOLOGY. 


131 


Tube  No.  8.  0.03  cc.  of  tube  B  mix- 
ture =  0.003  cc.  hemolytic  serum. 

Tube  No.  9.  0.02  cc.  of  tube  B  mix- 
ture —  0.002  cc.  hemolytic  serum. 

Tube  No.  10.  0.01  cc.  of  tube  B  mix- 
ture =  0.001  cc.  hemolytic  serum. 

5.  To  each  of  the  above  ten  tubes  add 

1  cc.  of  the  red  blood  cells  sus- 
pension. 

6.  Add  sufficient  normal  saline  to  the 

tubes  containing  a  small  amount 
of  material,  to  bring  the  columns 
of  fluid  to  the  same  level. 

7.  Shake  each  tube  so  as  to  thoroughly 

mix  its  contents.  Plug  the  mouth 
of  the  tube  with  cotton  and  place 
all  in  the  incubator  at  37°  C.  for 
1  hour. 

8.  Remove   the  tubes  from   the  incu- 

bator and  into  each  tube,  by 
means  of  a  pipette,  place  0.1  cc. 
complement  (guinea  pig  serum); 
replace  tubes  in  incubator  for  1 
hour. 

9.  Remove  the  tubes  from  the  incu- 

bator and  if  all  the  tubes  are  not 
completely  hemolized,  stand  on 
one  side,  in  ice-chest  if  possible, 
for  1  hour. 

10.  Examine  all  tubes  for — 
Complete  hemolysis  —  clear  red  so- 
lution, no  deposit  of  red  cells  at 
the  bottom  of  the  tube. 

Absence  of  hemolysis  —  clear  or 
turbid  colorless  fluid,  with  a  de- 
posit of  red  cells  at  the  bottom  of 
the  tube. 

The  smallest  amount  of  hemolytic 
serum  causing  complete  hemolysis  is 
known  as  the  minimal  hemolytic  dose 
(M.H.D.)  and,  if  hemolysis  has  oc- 
curred in  all  of  the  tubes  down  to 
No.  7,  the  M.H.D.  of  this  particular 
serum  is  0.005  cc.  200  minimal  hemo- 
lytic doses  per  cc.  This  serum  is 
strong  enough  for  experimental  work. 
As  a  matter  of  fact,  complete  hemo- 
lysis down  to  tube  No.  6  will  indicate 
a  serum  sufficiently  strong  (=  100 
M.H.D.  per  cc.)     If  the  first  one  or 

^  two  tubes  show  complete  hemolysis 
only,  the  rabbit  should  receive  further 
injections  in  order  to  raise  the  hemo- 
lytic power  to  the  proper  high  level. 


132  BACTERIOLOGY. 


THE  STORAGE  OF  HEMOLYSIN. 

If  the  rabbit  serum  hemolytic  contents 
is  found  to  be  sufficient,  chloroform 
the  rabbit  and  remove  aseptically  as 
much  blood  as  possible  from  the  heart 
and  place  it  in  sterile  centrifuge  tubes. 
Place  the  tubes  in  the  incubator  at 
37°  C.  for  2  hours,  after  which  times 
centrifugalize  thoroughly.  Pipette  off 
the  clear  serum  and  fill  in  quantities 
of  1  cc.  into  small  pipettes,  sealed 
hermatically,  in  the  blow  pipe  flame, 
avoid  scorching  the  serum.  Place  the 
small  pipette  containing  the  serum, 
after  having  been  sealed  in  the  water 
bath  at  56°  C.  for  30  minutes  (de- 
stroying the  complement) ;  i.  e.,  in- 
activating the  serum  and  at  the  same 
time  insuring  sterility.  A  longer  ex- 
posure reduces  the  hemolytic  power. 
Place  the  pipette  in  a  metal  box  and 
store  in  the  ice-chest. 

IjYSINS  are  substances  occurring  in 
normal  and  immune  sera  which  have 
the  power  of  destroying  and  dissolv- 
ing bacteria  and  dissolving  or  liberat- 
ing the  hemoglobin  of  the  red  blood 
cells  and  also  have  a  lytic  action  on 
the  various  body  cells. 

When  acting  on  bacteria,  they  are  called 
bacterolysins;  on  the  red  blood  cells, 
hemolysins;  on  the  body  cells,  cyto- 
lysins.  The  piechanism  of  the  process 
is  complex.  Certain  substances  which 
kill  bacteria  and  the  body  cells  but 
do  not  actually  dissolve  them  are 
spoken  of  respectively  as  bactericidal 
substances  and  cytotoxins. 

Blood  cells  of  one  animal,  injected  into 
another  animal  of  another  species, 
gives  rise  to  a  hemolytic  substance  in 
the  blood  serum  of  the  animal  in- 
jected, which  is  specific  for  the 
variety  of  cells  injected.  These  hemo- 
lysins are  termed  heterolysins. 

Ehrlich  and  Morgenroth  injected  the 
washed  red  blood  cells  of  one  goat 
into  another  and  found  that  the  serum 
of  the  injected  goat  would,  after  a 
time,  develop  hemolytic  power  against 
the  blood  cells  of  the  goat  whose 
blood  cells  had  been  used  but  did  not 
possess  hemolytic  power  toward  the 
red  blood  cells  of  all  goats.  Such 
substances   producing   hemolysins  in 


BACTERIOLOGY. 


133 


members  of  its  own  species  are  called 
isolysius. 

The  injection  of  isolysins  produced  anti- 
isolysins  which  were  again  specific. 
They  were  not  able  to  produce  sub- 
stances that  would  hemolyze  the  an- 
imals own  red  blood  cells  (autolysin). 

Lytic  substances  can  be  prepared  for  a 
large  number  of  bacteria  and  for 
many  body  cells.  These  bodies  may 
be  increased  markedly  during  the  pro- 
cess of  immunization.  The  substances 
having  the  power  to  produce  lysins 
are  called  lysinogen  and  are  distinct 
antigens  as  the  lysins  are  antibodies. 
The  lysins  may  be  prepared  by  in- 
jecting the  live  cells,  the  dead  cells, 
the  disintegration  products  of  cells 
and  in  some  cases  the  metabolic  pro- 
ducts of  cells. 

From  the  fact  that  the  bacteriolytic 
digestive  power  of  immune  serum 
after  being  destroyed  by  heating  or 
attenuated  by  time  can  be  restored — 
"reactivated" — by  the  addition  of 
small  quantities  of  normal  blood 
serum,  Borded  concluded  that  the 
bactericidal  or  bacteriolytic  action  of 
the  serum  depended  upon  two  sub- 
stances. One  present  in  normal  serum 
and  thermolabile,  he  identified  as 
Buchner's  alexin.  The  other,  more 
stable,  produced  or  increased  in  serum 

-  by  immunization,  he  called  the  "sensi- 
tizing substance,"  which  he  believed 

:  acted  upon  the  bacterial  cells  and 
rendered  them  susceptible  to  the  ac- 
tion of  alexin. 

Ehrlich  called  the  thermolable  substance 
or  alexin  "compliment"  and  showed 
that  it  was  always  present  in  normal 
serum  and  was  very  little,  if  at  all, 
increased  during  the  process  of  im- 
munization. 

The  "sensitizing  substance"  he  called 
the  "immune  body,"  and  this  he 
showed  to  be  increased  during  im- 
munization. Ehrlich  argued  that  when 
bacteria  or  blood  cells  were  injected 
into  the  animal,  certain  chemical 
components  of  the  injected  substances 
were  united  to  side  chains  of  proto- 
plasm of  the  tissue  cells.  The  ex- 
cessive production  of  these  receptors 
caused  their  detachment  and  subse- 
quent invasion  into  the  circulation  as 


134 


BACTERIOLOGY. 


"immune  bodies.'*  These  immune 
bodies  must  therefore  possess  atom 
complexis,  which  endow  it  with  chem- 
ical affinity  for  the  bacteria  or  red 
blood  cells  used  in  its  production. 

The  complement  does  not  combine  di- 
rectly with  the  blood  cell  or  bacteria, 
but  does  so  through  the  intervention 
of  the  immune  body  which  possesses 
two  atom  groups  or  haptophores;  one 
the  cytophile  haptophore  group,  pos- 
sessing strong  chemical  affinity  for 
the  blood  cell  or  bacteria;  the  other 
complementophile  haptophore  group- 
possessing  a  weaker  affinity  for  the 
•complement. 

Blood  cell  ,<rvc^  rmiTTTN  ^  


The  Structure  of  I^ysins. 

Ijysins  and  bactericidal  substances  are 
composed  of  a  thermolabile  part — 
"complement" — which  is  destroyed  at 
a  temperature  of  56°  to  60°  for  30 
minutes  and  a  thermolstable  part — 
"amboceptor" — having  a  double  com- 
bining power.  The  amboceptor  with- 
stands a  60°  C.  temperature  for  24 
hours,  but  will  be  destroyed  at  a  tem- 
perature of  70°  C.  Ehrlich  believes 
these  amboceptors  to  be  free  chemical 
receptors  of  the  body  cells  produced 
toy  the  same  method  as  are  the  anti- 
toxins, but  differing  from  them  in 
that  they  have  two  combining  groups; 
one  called  the  citophyle  group,  with 
which  the  amboceptor  combines  with 
bacteria  or  other  cells;  and  the  other, 
the  complementaphyle  group,  with 
which  it  combines  with  the  comple- 
ment. 

J  The  complement  seems  to  be  a  normal 
constituent  of  the  blood  sera  and 
other  body  fluids  and  is  undoubtedly 
produced  by  the  various  body  cells, 
leucocytes,  etc.  During  the  immuni- 
zation  of  animals,   it   increases  but 


or  , 


t  .  t 


^^Complement 


Cy  fopb\\C  Complemenlophilc 


Haptophore 


BACTERIOLOGY. 


135 


slightly,  if  at  all.  It  is  supposed  to 
be  made  up  of  two  groups,  one  a  hap- 
tophore  group,  which  combines  with 
the  amboceptor,  and  another  a  zyma- 
phore  group  which  really  produces  the 
lytic  action  after  the  haptophore  has 
combined  with  the  amboceptor.  On 
heating  the  complement,  the  zymo- 
phore group  is  destroyed  and  a  com- 
plementoid  is  produced.  This  sub- 
stance is  similar  to  a  toxid  and  will 
combine  with  amboceptor  but  no  lysis 
will  result.     (See  antitoxins). 

It  is  the  amboceptor  that  is  increased 
during  process  of  immunization.  The 
complement  will  not  combine  with  the 
cells  unless  the  amboceptor  is  present 
and  has  first  combined  with  the  cells. 
It  is  probable  that  in  the  body  fluids 
there  are  many  complements  which 
may  activate  a  variety  of  ambocep- 
tors. Various  sera  have  been  noted 
which  possess  amboceptors  for  cer- 
tain cells,  but  they  are  not  lytic  be- 
cause they  do  not  possess  the  neces- 
sary complement.  For  example,  dog 
serum  contains  amboceptors  for  the 
anthrax  bacterium,  but  no  comple- 
ment. If,  in  such  cases,  a  foreign 
complement,  such  as  guinea  pig  or 
rabbit  serum  is  added,  there  will  be 
lysis  of  the  bacterial  cells. 

Occassionally  the  absence  of  a  comple- 
ment may  benefit  an  animal,  and  this 
may  account  for  the  seeming  natural 
immunity  as  illustrated  when  the 
venom  (this  is  nothing  more  than  am- 
boceptors) of  a  poisonous  snake  is 
injected  into  an  animal,  such  as  the 
hog,  which  possesses  no  complement, 
no  lysis  of  the  cells  takes  place.  On 
the  other  hand,  should  the  animal, 
such  as  rabbit  or  man,  possess,  as 
they  do,  the  necessary  complement, 
lysis  will  take  place. 

Substances  are  sometimes  normally 
present  in  sera  which  have  the  power 
of  combining  with  amboceptors  which 
may  be  present  and  in  turn,  prevent 
the  amboceptors  from  combining  with 
the  cells  so  that  when  the  comple- 
ment is  added  there  will  be  no  lysis. 
These  substances  are  spoken  of  as 
anti  amboceptors  and  they  may  be  de- 
veloped in  an  animal  by  immuniza- 
tion with  amboceptors  of  definite  kind 


136  BACTERIOLOGY. 


(anti  antibodies).  Certain  other  sub- 
stances which  may  also  engage  the 
amboceptors  but  cannot  be  called  anti- 
amboceptors  in  the  true  sense,  ac- 
complish the  same  purpose  and  are 
therefore   classed   with   these  bodies. 

Deviation  of  the  Complement.  The  com- 
plement may  be  deviated  in  several 
ways,  and  as  a  result  lysis  may  be 
prevented.  Sometimes  there  is  noted 
in  sera  normal  substances  which  com- 
bine with  the  complement  and  prevent 
it  from  combining  with  the  ambo- 
ceptors. These  substances  are  spoken 
of  as  anti  complements  and  may  be 
produced  by  the  immuniation  of  an- 
imals with  complements. 

The  complement  may  occasionally  be 
absorbed  by  tissue  cells  and  in  this 
way  prevented  from  combining  with 
the  amboseptor.  In  certain  cases, 
where  the  serum  contains  an  excess 
of  amboceptors  and  only  a  small 
amount  of  complement,  it  may  be 
deviated  by  reason  of  the  fact  that  the 
cells  will  then  have  taken  up  all  the 
possible  amboceptors,  leaving  an 
abundance  of  free  amboceptors  in  the 
serum  to  combine  with  the  comple- 
ment, so  that  no  lysis  will  take  place. 
(This  theory  advanced  by  Neisser  and 
Wechsberg  has  created  wide  discus- 
sion but  is  believed  to  be  erroneous). 

Fixation  of  Complement  (as  a  test  for 
antibodies).  The  demonstration  of 
this  was  first  worked  out  by  Neisser 
and  Wechsberg  and  it  is  now  used  for 
testing  the  sera  for  unknown  anti- 
bodies similar  to  bactericidal  sub- 
stances and  lysins.  The  reaction  is 
made  use  of  in  the  recently  devised 
test  for  syphilis,  (See  Wasserman 
reaction). 

Filtration  of  Immune  Body  and  Comple- 
ment. 

Filtration  of  the  serum  will  allow  the 
amboceptor  to  pass  through  while  the 
complement  is  held  back.  The  am- 
boceptor filters  equally  well,  whether 
or  not  mixed  with  the  complement. 

COMPLFMENT  FIXATION. 
Test  for  Immune  Body  (Amboceptor). 

The  term  "complement  fixation"  is  ap- 
plied to  a  method  of  investigation  by 
which   even   the   small  quantities  of 


BACTERIOLOGY. 


137 


any  given  amboceptor  can  be  demon- 
strated in  the  serum.  The  method 
was  devised  by  Bordet  and  Gengrou 
and  prepared  as  follows: 


Tube  No.  1 

Bacterial  Ambo- 
cepto 

(Plague  immune 
serum,  heated). 

Plague  Emulsion. 

Complement 

(Fresh  Normal 
serum). 

— Allowed  to  act  for 


Hemolytic  ambo- 
ceptor 

(Heated  hemo- 
lytic serum). 

Red  blood  cells. 


Tube  No.  2 

Normal  serum, 
heated. 


Plague  Emulsion. 

Complement 

(Fresh  Normal 
serum). 

5  hours,  then  add — 

Hemolytic  ambo- 
ceptor. 

(Heated  hemo- 
lytic serum). 

Red  blood  cells. 


=  Results  = 


No  hemolysis.  Hemolysis. 

(Fluid  turbid  or  (Fluid  clear  and 
with  red  ppt).  red). 

The  conclusion  to  be  drawn  from  this, 
would  be,  that  in  No.  1,  the  presence 
of  amboceptor  had  led  to  the  absorp- 
tion of  all  the  complement  and  that 
in  No.  2,  there  being  no  bacteriolytic 
immune  body  to  sensitize  the  bacteria 
and  enable  them  absorb  complement, 
the  complement  was  therefore  left 
free  to  activate  the  added  hemolytic 
amboceptors. 

This  principle  of  complement  fixation, 
discovered  by  Boedet  and  Gengou  in 
1901,  has  been  utilized  in  bacteriolog- 
ical investigations  and  in  the  prac- 
tical diagnosis  for  the  determination 
of  specific  antibodies  in  serum. 

The  reaction  depends  upon  the  fact  that 
when  an  antigen  is  mixed  with  its 
inactivated  antiserum,  in  the  presence 
of  complement,  the  complement  Is 
firmly  fixed  by  the  combined  ambo- 
ceptor and  antigen  in  such  a  way  that 
it  can  no  longer  be  found  free  in  the 


138  BACTERIOLOGY. 


mixture.  If  this  mixture  is  now  al- 
lowed to  stand  at  a  suitable  tempera- 
ture for  an  hour  or  more,  then  an 
emulsion  of  red  blood  cells  and  in- 
activated hemolytic  serum  are  added, 
no  hemolysis  will  take  place  as  there 
is  no  free  complement  present  to  com- 
plete the  hemolytic  system.  (See  e.  g. 
below). 


-Complement 


^Hemolytic  )  Hemolytic  System 
Amboceptor  [  (Hemolysis) 


=Red  blood  celr 


N/1 


Immune  Body 
(Antibody) 


ZZ.  Antigen 


Eg.  =  Test  tube  containing—^ 
—  Complement. 


—  Patient's  serum  containing 
immune  body  (antibody). 

—  Antigen. 


Placed  in  incubator  at  37.5°  for 


one  hour,  then  add — 

— Hemolytic  ambocepto 
— Red  blood  cells. 
  =  Results  =   


BACTERIOLOGY.  139 

No  hemolysis  occurs  by  reason  of  the 

immune  body  combining  with  the 
complement  and  antigen — 


(I)       (+)  (5) 

Allowing   the   red   blood   cell   and  the 
hemolytic  amboceptor  to  remain  free — 


CI)  (2)  (3) 


No  completion  of  hemolytic  system. 

If  the  original  mixture  contains  no  anti- 
body for  the  antigen  used,  the  com- 
plement present  is  not  fixed  and  Willi 
be  available  for  the  activation  of  the 
hemolytic  serum.  (See  (1)  (2)  (3> 
above). 

The  reaction  is  therefore  seen  to  de- 
pend upon  the  fact  that  neither 
antigen  alone  nor  amboceptor  alone 
can  fix  the  complement,  but  that  this 
fixation  is  carried  out  only  by  the 
combination  of  antigen  plus  ambo- 
ceptors. Any  specific  amboceptor  can 
be  determined  by  this  method,  pro- 
vided the  hemologous  or  stimulating 
antigen  is  used  and  vice  versa. 

DETERMINATION  OF  ANTIBODIES 
BY  COMPLEMENT  FIXATION. 

When  testing  immune  sera  for  certain^ 
amboceptors  in  man  or  animals  by 
microorganisms  which  can  be  culti- 
vated, either  the  whole  organism  or- 
its  extracts  may  be  used  as  antigen.. 

Bordet  and  Gengou  use  a  thick  6alt  so- 
lution emulsion  of  a  24  hour  agar 
slant  culture  of  the  organism.  In  the 
use  of  tubercle  bacilli,  80  mg.  ot 
bacilli  are  emulsified  in  1  cc.  of  salt 
solution. 

Wasserman  and  Bruck  prepare  antigen 
by  emulsifying  10  agar  slant  cul- 
tures in  10  cc.  of  sterile  distilled 
water,  after  which  it  is  placed  in  a 


140  BACTERIOLOGY. 


shaking  apparatus  and  shaken  for  24 
hours.  0;  5%  of  carbolic  acid  is  added 
and  the  fluid  cleared  by  centrifuga- 
tion.  The  old  or  the  new  tuberculins 
or  "Bacillary  Emulsion"  are  used. 
Method. 

H.  S.  =  Hemolytic  serum  (heated  for 
15  min.  at  56°  C,  i.  e.  inactivated). 

Comp.  =  Complement  (Fresh  guinea 
pig  serum). 

H.  R.  B.  C.  =  Human  red  blood  cells. 

S.  S.  —  Specific  serum  from  inocu- 
lated animals,  —  inactivated. 

P.  S.  =  Control  "pooled  serum"  from 
normal  animals  of  the  same  species, 
—  inactivated. 

Ant.  =  Antigen  (organism  grown  on 
solid  media  and  previously  having 
served  as  antigen  in  the  inoculated 
animals). 

 Place  in  test  tubes  

12  3 


0.1  cc.  Comp.    —   0.1  cc.  Comp. 

0.2  cc.  S.  S.       0.2  cc.  S  S.  

1.0  cc.  Ant.       1.0  cc.  Ant.       1.0  cc.  Ant. 

4  5 


0.1  cc.  Comp.     0.1  cc.  Comp. 

0.2  cc.  P.  S.   

0.1  cc.  Ant.   

 Incubate  at  37°  C.  for  one  hour  

Add  to  each  tube  1  cc.  of  H.  R.  B.  C. 

and  4  minimal  hemolytic  doses  (see 
titration  of  hemolytic  serum)  of  H.  S. 

 Incubate  at  37°  C.  for  one  hour  

Results 

No.  1  —  No  hemolysis  =  indicates  the 
presence  in  the  serum  of  the  in- 
oculated animal  of  a  specific  anti- 
body to  the  organism  used  in  the 
inoculations;  since  it  shows  that 
the  complement  has  been  bound  by 
the  immune  body  to  the  bacterial 
antigen  and  none  has  been  left  free 
to  enter  into  the  hemolytic  system. 

Hemolysis  indicated  that  no  appreci- 
able amount  of  antibody  has  yet 
been  formed  in  response  to  the  in- 


BACTERIOLOGY. 


141 


oculations;  i.  e.,  there  is  no  infec- 
tion, since  the  complement  remained 
unfixed  at  the  time  of  the  addition 
of  the  H.  R.  B.  C.  solution  and  the 
H  S 

No.  2  =  No  hemolysis  ( 

No.  3,  4,  5  =  Hemolysis  V''°lt°  No.  1. 

It  may  sometimes  be  convenient  to 
sensitize  the  H.  R.  B.  C.  just  be- 
fore they  are  needed.  This  is  done 
before  the  completion  of  the  first 
period  of  incubation. 
Method. 

Place  5  cc.  of  H.  R.  B.  Q.  and  20  min- 
imal doses  of  H.  S.  in  a  sterile  test 
tube  and  allow  them  to  remain  at 
room  temperature  for  15  minutes. 
The  red  cells  are  then  sensitized 
and  ready  for  use. 

When  the  tubes  are  removed  from 
the  incubator  at  the  end  of  the  first 
hour,  1  cc.  of  the  sensitized  red 
cells  are  added  to  each  tube;  mixed 
thoroughly  and  the  tubes  returned 
to  the  incubator  for  the  second 
period. 

Complement  Fixation  for  the  Determ- 
ination of  Immune  Body  of  Syphilis. 

The   so   called   "Wasserman  Reaction" 

(although  not  strictly  belonging  to 
the  domain  of  bacteriology)  has  re- 
cently become  so  prominent  as  a 
diagnostic  agent  in  syphilis  that  an 
outline  of  it  will  be  given. 
1.  Antigfen. 

Wasserman  first  made  use  of  salt 
solution  extracts  of  the  organs 
(spleen,  etc.)  of  a  syphilitic  fetus, 
in  which  the  uncombined  products 
(free  syphilitic  antigens)  of  spiro- 
chaete  pallida  were  assumed  to  be 
present.  He  cut  the  tissue  into 
small  pieces  and  added  4  parts  by 
weight  of  a  normal  salt  solution 
containing  0.5%  of  carbolic  acid  to 
1  part  of  the  tissue.  This  was 
placed  in  the  shaking  apparatus  for 
24  hours  and  then  centrifugalized. 
The  supernatent  liquid  was  used  as 
the  antigen.  Porges  and  Meyer  pre- 
pared antigen  by  extracting  syphi- 
litic organs  with  alcohol.  The 
syphilitic  liver  was  chopped  up  and 
extracted  with  5  volumes  of  abso- 
lute alcohol  for  24  hours,  filtered 


1^2  BACTERIOLOGY. 

through  paper  and  then  the  alcohol 
was  evaporated  in  vacuo  at  a  tem- 
perature not  exceeding  40°  C.  About 
1  gm.  of  the  greenish  residue  is 
emulsified  in  100  cc.  of  salt  solu- 
tion containing  0.5%  of  carbolic 
acid  and  filtered  through  thin  paper. 
The  filtrate  is  used  as  the  antigen. 

Nogruchi  prepared  antigen  by-  thor- 
oughly macerating  normal  liver  or 
spleen  in  five  times  its  volume  of 
absolute  alcohol.  Place  it  in  the 
incubator  and  allow  it  to  extract 
for  6  to  8  days  with  thorough  stir- 
ring daily.  It  is  then  passed 
through  cheese  cloth  and  filtered 
through  paper.  The  extract  is  now 
evaporated  to  dryness  at  room  tem- 
perature and  the  sticky,  brownish 
residue  is  dissolved  in  a  small 
quantity  of  ether  and  4  times  its 
volume  C.  P.  acetone  is  added.  A 
heavy,  sticky,  brown  precipitate 
settles  to  the  bottom.  This  mass 
is  used  as  the  antigen  and  may  be 
preserved  under  acetone.  The  ace- 
tone soluble  fraction  is  discarded. 
When  wanted  for  use,  about  0.2 
gm.  of  the  mass  is  dissolved  in 
about  5  cc.  of  ether,  100  cc.  of 
normal  salt  solution  added  and 
shaken  till  the  ether  has  evap- 
orated. The  antigen  is  now  titrated, 
and  when  this  is  accomplished  it 
is  ready  for  use. 

The  use  of  syphilitic  organs  for  the 
preparation  of  antigen  is  not  neces- 
sary in  order  to  obtain  a  subtsance 
which  will  combine  with  the  syphi- 
litic immune  body. 

Many  nonspecific  antigens  will  give 
reasonably  reliable  results.  Porges 
and  Meier  found  that  a  1%  com- 
mercial lecithin  in  carbolized  salt 
solution  furnished  a  suitable  anti- 
gen. This  has,  however,  not  been 
universally  accepted. 

They  also  found  that  normal  foetal 
liver  would  give  good  results;  like- 
wise others  have  successfully  used 
an  alcoholic  extract  of  guinea  pig 
heart. 

The  ingredient  furnishing  the  immune 
body  binding  power  is  unknown  as 
yet,  although  it  is  claimed  to  be 
due  to  the  lipoids. 


BACTERIOLOGY. 


143 


Antigen  must  be  standard  before  It 
can  be  used  for  the  actual  test. 
The  substances  used  as  antigens 
often  have  the  power,  if  used  in 
too  large  quantities  of  binding  the 
complement.  It  is  therefore  neces- 
sary to  determine  the  largest  quan- 
tity of  antigen  which  may  be  used 
without  binding  complement.  This 
may  be  done  by  mixing  graded 
quantities  of  antigen  with  a  con- 
stant quantity  of  complement,  in 
duplicate  sets,  and  adding  to  each 
tube  of  one  set  0.2  cc.  of  a  normal 
serum  and  to  the  other  0.2  cc.  of  a 
known  syphilitis  serum.  These 
substances  are  allowed  to  remain 
together  for  one  hour  and  then  red 
blood  cells  and  inactivated  hemo- 
lytic serum  are  added.  The  quan- 
tity which  has  caused  complete  in- 
hibition with  the  syphilitic  serum, 
but  none  with  normal  serum,  is  the 
one  to  be  used  in  the  subsequent 
tests. 

A  dilution  of  the  antigen  should  be 
made  with  salt  solution  in  such 
a  way  that  1  cc.  shall  contain  the 
required  amount  of  antigen.  (E.  g. 
if  0.05  cc  is  wanted,  mix  0.5  cc.  with 
9.5  cc.  of  salt  solution.  1  cc.  of 
this  can  be  used  in  each  tube  in  the 
test). 

The   Hemolytic   Semm,  Amboceptor. 

Prepared  as  outlined  on  page  130, 
using  sheep's  blood. 
By  reason  of  the  fact  that  small 
amounts  of  precipitins  for  sheep's 
serum  may  be  present  in  the  serum, 
due  to  insufficient  washing  of  the 
corpuscles  employed  in  the  immuni- 
zation, and  cause  the  formation  of 
precititates  which  have  a  tendency 
to  carry  down  the  complement  from 
a  mixture,  it  is  therefore  necessary 
that  the  serum  is  of  high  potency 
in  order  that  the  quantities  used 
for  the  reaction  may  be  as  small  as 
possible. 

The  smallest  amount  of  hemolytic 
serum  that  has  caused  complete 
hemolysis  in  1  cc.  of  a  5%  emul- 
sion of  washed  blood  corpuscles  is 
spoken  of  as  the  hemoljrtic  nnlt. 

Many  make  use  of  two  hemolytic 
units  for  the  actual  reaction. 


144  BACTERIOLOGY. 


Complement  (fresh  guinea  pig  serum) 
is  obtained  by  anesthetizing  a 
guinea  pig,  incise  the  carotid  artery 
and  allowing  the  blood  to  flow  into 
a  large  Petri  dish.  The  dish  is  put 
away  in  the  ice  chest  until  the 
serum  has  separated,  which  is  then 
carefully  removed. 

The  serum  may  be  centrifugized  to 
insure  complete  separation  from 
blood  cells.  The  complement  in 
G.  P.  S.  is  for  practical  purposes, 
constant  in  quantity.  It  should  be 
kept,  except  when  in  actual  use,  or 
a  low  temperature,  and  should  not 
be  used  after  3  days  from  the  time 
of  preparation. 

Sheep's  Corpuscles  are  obtained  by 
receiving  the  blood  of  a  sheep  in — 

(1)  a  flask  containing  glass  beads, 
and  shaking  thoroughly  for  about 
10  minutes  to  completely  defibrin- 
ate  the  blood.  The  corpuscles  are 
washed  free  from  the  serum  by 
centrifugalization    in    salt  solution 

(2)  a  flask  containing  a  sterile  solu- 
or 

tion  of  0.5  %  sodium  citrate  and 
0.85%  sodium  chloride,  which  will 
prevent  clotting  and  the  corpuscles 
may  be  washed  free  from  the 
citrate  solution  by  centrifugaliza- 
tion in  salt  solution.  Thorough 
washing  with  salt  solution  is  essen- 
tial in  order  to  preclude  the  oc- 
curence of  precipitates  and  to  re- 
move traces  of  complement.  The 
bulk  of  the  centrifugalized  cor- 
puscles is  measured  and  19  parts  of 
sterile  salt  solution  is  added.  This 
forms  a  5%  enlulsion  of  corpuscles 
which  is  the  solution  employed  for 
the  test. 

Serum  to  toe  tested  for  Syphilitic  anti- 
body. 

3  to  5  cc.  of  blood  is  removed  from  a 
vein,  or  if  conditions  will  not  per- 
mit such  procedure,  the  blood  may 
be  obtained  from  the  finger  or  ear. 
The  quantity  of  blood  must  always 
be  of  suflJicient  quantity  to  furnish 
1  cc.  of  clear  serum.  The  serum  is 
now  inactivated  by  heating  at  56° 
C.  for  20  to  30  minutes. 

Noguchi  advises  inactivation  at  54°  C. 
as  the  56°  C.  destroys  the  syphilitic 
antibody  in  part. 


BACTERIOLOGY. 


145 


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146  BACTERIOLOGY. 


 Place  in  water  bath  at  40*  C.  for 

1  hour  

Add  to  each  tube  1  cc.  of  a  5%  solution 
of  sheeps'  corpuscles  and  two  units 
of  amboceptor. 

 Place  in  water  bath  at  40**  'C.  for 

1  to  2  hours  

Xtesnlts. 

If  test  is  positive,  tubes  3A  and  4A 
will  show  no  liemolysis  while  all 
other  tubes  show  complete  hemo- 
lysis. 

Tube  No.  1.  "A"  shows  active  comple- 
ment. "B"  shows  antigen  alone  is 
not  sufficient  to  deviate  comple- 
ment. 

Tube  No.  2.  "A"  shows  no  deviation  of 
complement  in  presence  of  normal 
serum  alone.  "B"  shows  that  the 
particular  serum  alone  will  not 
deviate  the  complement. 

Tube  No.  3.  "A"  shows  that  antigen 
is  specific  in  that  it  deviates  com- 
plement in  the  presence  of  syphi- 
litic antibody.  "B"  shows  syphi- 
litic antibody  will  not  alone  deviate 
the  complement. 

Tube  No.  4.    "A"  shows  the  presence 

.  of  an  antibody  specific  to  the  anti- 
gen employed  in  that  the  comple- 
ment was  deviated  by  antigen  in 
presence  of  test  serum. 
Nogruchi's  Modification  of  the  Wasser- 
man  Test. 

Anti-human  hemolytic  amboceptor  is 
used  instead  of  an  anti-sheep  am- 
boceptor. It  is  obtained  by  4  or  5 
injections  of  washed  human  cor- 
puscles into  rabbits.  The  ambo- 
ceptor unit  is  obtained  as  in  the 
original  Wasserman.  Two  units  are 
used. 

The  fact  that  human  serum  contains 
normally  no  amboceptor  active 
against  the  human  red  corpuscle  is 
important  and  has  an  advantage 
over  the  original  Wasserman. 

Human  serum,  normally,  may  contain 
a  variable  quantity  of  amboceptor 
for  sheep's  corpuscles,  consequent- 
ly the  actual  amount  of  hemolytic 
amboceptor  used  in  the  original 
Wasserman  is  uncertain.  This  is 
not  so  in  the  Noguchi,  as  the  actual 
quantity  of  amboceptor  is  known 
exactly  by  titration. 


BACTERIOLOGY. 


147 


Antig'en.    Prepared  as  in  Wasserman. 

Complement.  A  40%  fresh  guinea  pig 
serum  is  made  by  mixing  1  part  of 
serum  with  1.5  parts  of  salt  solu- 
tion. 0.1  cc.  of  this  solution  is  used 
for  the  test. 

Siiman  Corpuscles.  Normal  corpus- 
cles, or  those  of  the  patient  himself, 
may  be  employed.  The  patient's 
corpuscles  should  not  be  used  for 
other  tests  than  that  on  the 
patient's  own  serum.  1  cc.  of  a  1% 
emulsion  of  washed  corpuscles  is 
used  for  the  test. 
Patient's  Serum  to  be  tested  for  the 
Syphilitic  antibody. 

Obtained  as  in  Wasserman  or  in 
Wright's  tube* 

About  2  cc.  should  be  taken. 


148 


BACTERIOLOGY. 


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BACTERIOLOGY.  149 


 Shake,  and  place  in  water  bath  at 

38°-40°  for  one  hour  

Add  to  each  tube  two  units  of  ambocep- 
tor and  the  human  red  blood  cell 
emulsion. 

— ^Shake,  and  replace  in  water  bath  for 
one  hour  or  more  till  controls  are 
hemolized  

BesTilts. 

If  test  is  positive  there  will  be  no 
hemolysis  in  tubes  lA  and  2A  while 
all  others  are  hemolized. 
Determinatiou  of  Autig'en  by  Comple- 
ment Fixation. 

In  testing  for  suspected  antigen,  the 
procedure  is  reverse  to  that  of  test- 
ing for  suspected  antibodies.  The 
serum  or  bacterial  extract  to  be 
tested  for  antigen  is  brought  into 
contact  with  an  antibody  specific 
for  the  antigen  in  the  presence  of 
complement;  and  at  the  end  of  an 
hour  at  suitable  temperature,  free 
complement  is  again  determined  by 
hemolytic  reaction,  as  in  the  anti- 
body tests. 

Hemolytic  Amboceptor.  Prepared  in 
the  rabbit  for  sheep  corpuscles. 

Inactivated  and  titrated  as  for  Was- 
serman  test. 

Two  units  are  used. 

Bacterial  Antiserum.  Prepared  by 
immunizing  a  rabbit.  It  must  be 
highly  potent.  The  smallest  quan- 
tity of  the  immune  serum  which 
will  fix  the  complement  in  the 
presence  of  an  emulsion  or  extract 
of  the  microorganism  in  question  is 
determined  by  experiment. 

The  bacterial  emulsion  is  prepared  by 
scraping  the  growth  from  24  hour 
agar  slant  cultures,  drying  it,  and 
macerating  in  a  mortar  with  salt 
solution  until  a  slight  opalescent 
emulsion  is  formed. 

Prepare  a  series  of  tubes,  each  con- 
taining 0.1  cc.  of  the  bacterial  emul- 
sion, 0.1  cc.  complement  and  gradu- 
ally diminishing  quantities  of  in- 
activated specific  immune  serum, 
ranging  from  0.1  cc.  downward. 

Incubate  the  tubes  at  38°-40°  C.  for 
1  hour. 

Add  the  required  quantities  of  red 
blood  cells  and  hemolytic  immune 
serum.     The   smallest   quantity  of 


150  BACTERIOLOGY. 


immune  serum  which  has  complete- 
ly inhibited  hemolysis  is  the  unit. 
A  quantity  slig-htly  in  excess  of  the 
unit  is  used  in  the  test. 

Complement.  Fresh  guinea  pigr  serum 
(0.1  cc.  used  in  routine  work).  'It 
should  however  be  titrated  if  pos- 
sible and  used  in  double  the  quan- 
tity necessary  to  produce  hemolysis 
of  1  cc.  of  a  5%  emulsion  of  blood 
cells,  in  the  presence  of  two  units 
of  amboceptor. 

Sheep  Corpuscles.  Prepared  as  in 
Wasserman  test. 

Patient's    Serum.     Obtained    by  the 
usual    method    and    inactivated  at 
56"  C.  for  20  minutes. 
Test. 

Prepare  a  series  of  tubes,  each  con- 
taining: 

1.  Complement,  0.1  cc.  or  the  deter- 

mined quantity. 

2.  Antiserum,    the    determined  quan- 

tity. 

3.  Serum  to  be  tested  for  antigen  in 

diminishing  quantities  from  1  cc. 
downward. 

4.  Salt  solution  for  dilution  to  3  cc. 

5.  Control  tubes  containing  the  same 

ingredients  without  the  antiserum. 

6.  Incubate  for  1  hour  at  40°  C. 

7.  Add  required  quantities  of  ambo- 

ceptor and  red  cells. 

8.  Incubate  again. 

Results  =  A  positive  reaction  if  there 
is  no  hemolysis  in  the  tubes  con- 
taining the  patient's  serum. 

Proteid  Differentiation  by  Complement 
Fixation,  such  as  human  or  animal 
blood  was  shown  by  Gengou  in  1902. 
The  test  is  said  to  be  more  delicate 
and  reliable  than  the  precipitation 
tests. 

Hemolytic  amboceptor.  Prepared  as 
for  Wasserman  test. 

Complement.  Prepared  as  for  Wasser- 
man test. 

Sheep  Corpuscles.  Prepared  as  for 
Wasserman  test. 

Specific  Antiserum.  Prepared  by  im- 
munizing a  rabbit  with  the  proteid 
material  for  which  the  test  is  to 
be  made.  .Filtrate  by  using  dim- 
inishing quantities  of  the  antiserum 
in  a  series  of  test  tubes  containg 
the    determined    quantity    of  com- 


BACTERIOLOGY.  151 


plement,  and  the  antigen  which  is 

to  be  tested  for,  i.  e.,  the  holologrous 

serum   with    which   the  antiserum 

has  been  produced. 
The  test  should  be  so  delicate  as  to 

determine  0.0001  cc.  of  the  antigen, 

consequently  this  quantity  is  added 

to  each  tube. 
The  tubes  are  incubated  for  1  hour. 

The  hemolytic  amboceptor  and  red 

cells  are  then  added. 
The    unit    represents    the  smallest 

quantity   of   antiserum   which  has 

completely  inhibited  hemolysis. 
One  and  one-half   to  two   units  are 

used  for  the  test. 
Solution  of  th.e  proteid  material  to  be 

tested.     Prepare   as   for  precipitin 

test. 
Test. 

Prepare  a  series  of  tubes  containing: 

1.  Complement,    quantity  determined 

by  titration. 

2.  Antiserum,  quantity  determined  by 

titration. 

3.  Diminishing  quantities  of  the  sub- 

stance in  which  the  antigen  is 
suspected,  ranging  from  0.1  cc. 
downward  to  0.0001  cc. 

4.  Salt  solution  to  make  dilution  to 

3  cc. 

5.  Control  tubes  containing  the  same 

ingredients  without  the  antiserum. 

6.  Incubate  for  1  hour  at  40°  C. 

7.  Add  j^equired  quantities  of  ambo- 

ceptor and  red  cells. 

8.  Incubate  again. 

Results  =:  A  positive  reaction  if  there 
is  no  hemolysis  in  the  tubes  con- 
taining the  suspected  antigen. 

EHBI^ICH'S  SIDE-CHAIN  THEORY  de- 
rives its  name  from  its  analogy  to 
what  happens  in  the  Benzol  ring  when 
its  replaceable  hydrogen  atoms  are 
substituted  by  "side-chains." 

The  theory  itself  is  based  upon  the 
mechanism  of  cell  nutrition  in  its  re- 
lation to  the  mode  of  production  of 
specific  antitoxins. 

In  order  that  a  cell  may  be  nourished, 
the  nutritive  substance  must  enter 
directly  into  chemical  combination 
with  some  elements  of  the  cell  pro- 
toplasm. 

The  highly  complex  protoplasmic  mole- 
cules of  cells  are  made  up  of  a  central 


152  BACTERIOLOGY. 


atom-group,  upon  which  the  special- 
ized activities  of  the  cell  depend,  and 
several  outer  atom-groups  (side- 
chains)  by  which  the  cell  entered  into 
chemical  relation  with  food  and  other 
substances  brought  to  it  by  the  cir- 
culation. 

In  just  the  same  way  the  nutritious  sub- 
stances are  brought  into  relation  with 
the  cell  by  means  of  the  side  atom- 
groups,  so  will  also  isomeric  toxins. 

These  side-chains  are  called  "receptors," 
and  if  they  have  an  affinity,  by  reason 
of  isomerism  or  chance  for  a  given 
toxin  they  unite  with  the  toxin  and 
are  tltferefore  rendered  useless  for 
their  normal  physiological  function  of 
nutrition. 

These  receptors  are  probably  cast  off 
and  regenerated  by  the  normal  re- 
parative mechanism  of  the  body. 

The  regenerative  process  does  not  stop 
at  simple  replacement  of  the  cast  off 
elements,  but  goes  on  to  over  compen- 
sation, so  that  they  are  reproduced 
in  excess  of  the  physiologic  needs  of 
the  cell,  and  therefore  cast  of£  to  cir- 
culate in  the  blood  as  antitoxins. 
These  receptors  (outer-atom-.groups, 
antitoxins)  retain  their  specific  affin- 
ity for  the  toxins  used  in  their  pro- 
duction and  will  unite  with  the  poison 
before  it  can  reach  the  sensitive  cells, 
and  in  this  way  protect  the  cell  from 
the  poison.  ^ 


Toxin  analysis  (Ehrlich). 

Toxin  solutions  deteriorate  with  time; 
i.  e.  a  toxin  bouillon  which  contained 
80  toxin  units  per  1  cc.  was  found  to 
contain  but  40  units  after  4  or  5 
months. 

Ehrlich  found  that  such  bouillon  re- 
tained its  full  original  power  ot 
neutralizing  antitoxin. 


BACTERIOLOGY. 


153 


The  toxin  molecule  must  therefore  con- 
tain two  separate  atom-groups.  One 
stable-group,  possessing-  the  power  of 
binding  antitoxin,  he  called  the  "hap- 
tophore"  or  "anchoring"  group.  The 
other,  the  one  by  which  the  toxin 
molecule  exerts  its  deleterious  action, 
is  more  easily  changed  or  destroyed, 
he  calls  the  "toxophore,"  or  poison 
group. 


In  the  toxin-bouillon  in  which  a  part  of 
its  poison  has  been  lost  while  the 
neutralizing  antitoxin  power  still  re- 
mains, it  is  quite  evident  that  the 
toxophore  group,  or  some  of  the  toxin, 
must  have  been  changed  or  destroyed. 
Altered  in  this  way  he  calls  it 
"toxoid." 

Substances  found  in  fresh  bouillon, 
which  have  a  weaker  affinity  for  anti- 
toxin than  toxin  itself,  called  "toxins," 
are  primary  secretory  products  of  the 
bacteria. 

The  "toxoids"  are  of  two  kinds — namely, 
those  which  have  a  stronger  affinity 
for  antitoxin  than  toxin  itself  (pro- 
toxoids),  and  those  whose  affinity  for 
antitoxin  is  equal  to  that  of  toxin 
(syntoxoids). 

The  toxon  has  a  haptophore  group  sim- 
ilar to  that  of  toxin,  but  a  different 
toxophore  group.  It  differs  from 
toxin  in  that  it  lacks  the  power  to 
produce  acute  symptoms;  it  causes 
gradual  emaciation  and  paresis  in  an- 
imals. 

The  toxophore  group,  producing  the 
harmful  results,  is  divided  into  toxin, 
toxoid  and  toxon. 


154  BACTERIOLOGY. 


The  haptophore  group  of  the  toxin, 
then,  possesses  the  affinity  for  the  re- 
ceptor or  antitoxin. 

The  haptophore  groups  of  all  three  of 
these  substances  are  alike.  In  toxid, 
the  toxophore  group  has  been  de- 
stroyed or  altered;  in  toxon  it  is 
qualitatively  different  from  that  of 
toxin.  It  should  therefore  produce 
antitoxins. 

AGG-IiUTIITINS. 

While  investigating  the  Pfeiffer  reac- 
tion with  B.  coli,  Gruber  and  Durham 
noticed  that  if  the  immune  serum 
was  added  to  bouillon  cultures  of  B. 
coli,  the  cultures  would  loose  their 
turbidity  and  flake-like  clumps  would 
sink  to  the  bottom  of  the  tube. 

Widal  applied  this  agglutination  reac- 
tion to  the  practical  diagnosis  of 
typhoid. 

Gruber  and  Durham  believed  the  ag- 
glutinins to  be  identical  with  the  im- 
mune body  concerned  in  the  Pfeiffer 
reaction,  which  injured  the  bacteria, 
thereby  rendering  them  susceptible  to 
alexins.  It  has  since  been  shown  that 
agglutinins  and  bactericidal  sub- 
stances are  in  no  way  parallel. 
Strongly  agglutinating  sera  may  be 
very  weak  in  bactericidal  power  and 
strongly  agglutinating  sera  may  be 
very  weak  in  agglutinating  power;  the 
relative  quantity  of  these  substances 
depends  upon  the  method  of  immuni- 
zation. 

Agglutinated  bacteria  are  not  killed  by 
the  algglutination  and  are  often  as 
virulent  as  non-agglutinated  cultures. 
Agglutinins  remain  active  after  ex- 
posure to  over  55°  C.  temperature. 
Some  will  withstand  65°  to  70*  C. 
and  can  not  be  reactivated  by  the  ad- 
dition of  normal  sera.  This  excludes 
the  participation  of  complement  in 
this  reaction. 

The  agglutinins  do  not  dialyze. 

Normal  sera  contain  small  amounts  of 
agglutinin  —  "normal  agglutinins"  — 
probably  due  to  the  various  micro- 
organisms parasitic  upon  the  animal 
body. 

Agglutinins  can  be  produced  by  intro- 
ducing microorganisms  subcutaneous- 
ly,  intravenously  or  intraperitoneally. 
Almost  all   the  known  bacteria  will 


BACTERIOLOGY. 


155 


produce  agglutinin,  and  it  will,  as  a 
rule,  appear  in  the  blood  of  animals 
three  to  six  days  after  the  micro- 
organisms' introduction;  increase  to 
a  maxim  at  the  7th  to  the  13th  day, 
they  then  fall  oft  till  they  reach  a 
level,  at  which  they  remain  for  a  long 
time. 

Agglutination  is  not  limited  to  bacteria; 
just  as  hemolysins  are  produced  by 
the  injection  of  red  blood  cells,  so 
hemagglutins  are  similarly  formed. 

Ag-grltitiuating'  Test. 
Microscopic  Method. 

1.  Collect  a  small  amount  (5-10  drops) 

of  blood  in  a  small  glass  tube. 

2.  Separate    the    serum   by  centrifu- 

gation. 

3.  By    means    of   Wright's  capillary 

pipette  or  the  white  mixing  pi- 
pette accompanying  the  hemocy- 
tometric  counting  chamber,  dilute 
the  serum  with  normal  salt  solu- 
tion in  proportion  of  1  to  20. 
From  this,  subdilute  in  propor- 
tion of  1-40,  1-80,  1-160,  etc.,  and 
place  each  dilution  in  a  separate 
sterile  watch  glass. 
(Dilutions  can  also  be  made  by  the 
drop  method,  using  a  capillary 
pipette  from  which  a  drop  of 
serum  is  placed  in  a  watch  glass 
and  then  normal  saline  dropped 
into  it  till  proper  dilution  is  ob- 
tained). 

4.  With   the   platinum   loop,   place  a 

drop  of  the  serum  from  each  of 
the  dilutions  of  serum  on  a  cover 
glass,  and  inoculate  each  with  a 
loopful  of  a  24  hour  old  bouillon 
growth  of  the  organism. 

5.  Press  the  cover  slips  carefully  over 

the  chamber  of  culture  slides,  the 
margins    of    v/hich    have  been 
singed    with    vaseline  (hanging 
drop  method). 
See  that  the  various  dilutions  are 
properly  indicated  on  the  slides. 
6.  Examine  immediately  under  the  1/6 
objective  of  the  microscope,  and 
discard  the  slides  if  any  clumps 
are   observed.     If   the   mount  is 
satisfactory. 
7.  Set  the  mount  aside  and  re-examine 
at  the  end  of  y2  hour.    If  reaction 
is   positive,   all   the  microorgan- 


156  BACTERIOLOGY. 


isms  will  be  found  motionless  and 
gathered  in  clumps  of  variable 
size. 

This  will  be  the  case  at  least  in  the 
lowest  dilutions,  while  in  the 
hig-her  ones  it  may  be  necessary 
to  wait  until  another  half  hour 
has  expired. 

The   hig-her   the   dilution   in  which 
complete    clumping    is  obtained, 
the    greater    is    the  diagnostic 
value. 
Macroscopic  method. 

This  test  is  made  in  series  of  small  test 
tubes  of  0.5x5  cm.  size.  In  these  test 
tubes  1  cc.  of  the  different  serum  dilu- 
tions and  the  bacterial  emulsion  are 
mixed.  They  are  now  placed  in  the 
incubator  for  a  few  hours  and  then 
kept  at  room  temperature. 

Hiss  has  observed  that  agglutination 
will  be  hastened  in  some  instances  if 
after  their  removal  from  the  in- 
cubator, they  are  placed  in  the  ice 
chest.  ^ 

Vv^hen  agglutination  takes  place,  clumps 
of  bacteria  are  seen  to  form,  which 
settle  to  the  bottom,  much  like  snow 
tfakes  on  the  tube.  The  surface  of 
the  sediment  is  heaped  up  and  irreg- 
ular. The  supernatant  fluid  becomes 
entirely  clear. 

When  the  reaction  is  negative,  the  sedi- 
ment is  an  even,  granular  one  with 
flat  surface,  and  the  emulsion  remains 
turbid. 


PRECIPITINS. 

Kraus  ,(1897)  demonstrated  that  sera  of 
animals  immunized  against  a  partic- 
ular microorganism  when  mixed  with 
the  clear  filtrate  of  bouillon  culture 
of  the  particular  organism,  would  give 
rise  to  visible  precipitate.  In  that  the 
precipitate  occurred  only  when  the 
filtrate  of  a  bouillon  culture  of  an 
organism  and  the  sera  of  animals 
immunized  with  same  organism  led 
Kraus  to  name  them  "specific  precipi- 
tates," or  precipitin. 

Precipitin  formation  is  not  limited  to 
bacterial  immunization,  but  is  also 
found  like  the  phenomena  of  agglut- 
ination and  lysis.    Substances  produc- 


BACTERIOLOGY. 


157 


ing-  the  phenomena  in  sera  are  called 
precipitinogens. 

Precipitins  like  agglutinins  may  be  in- 
activated by  heating  to  from  60°  to 
70°  C.  and  cannot  be  reactivated  by 
adding  normal  sera,  etc.  Inactivated 
precipiptin,  while  unable  to  produce 
precipitates,  will  bind  the  precipi- 
tinogen. This  is  shown  when  inacti- 
vated precipitin  is  mixed  with  pre- 
cipitinogen no  reaction  occurs  if  fresh 
precipitin  is  added. 

Precipitin  is,  therefore,  like  toxin,  made 
up  of  two  atom-g-roups,  a  stable  hap- 
tophore  and  a  labile  precipitophore 
group.  It  is  the  opinion  of  many  that 
precipitins  are  identical  in  structure 
with  amboceptors.  Just  as  in  ag- 
glutins  there  is  in  precipitin  a  certain 
degree  of  "group  reaction";  that  is, 
the  precipitin  obtained  with  a  colon 
bacillus  will  cause  a  precipitation 
with  culture  filtrates  of  allied  organ- 
isms. This  may  be  easily  adjusted, 
however,  by  the  use  of  proper  dilu- 
tion similar  to  that  used  in  ag-g-lutina- 
tion  tests. 

Wasserman  and  others  found  other  lise 
for  this  reaction  as  a  means  of  dis- 
tinguishing the  blood  of  one  species 
from  that  of  another.  Precipitins 
have  not  been  demonstrated  in  normal 
sera. 

PRECIPITIN  TESTS. 

These  tests  jnay  not  only  be  applied  to 
bacteria,    but    also    to    the  various 
proteid  substances. 
Bacterial  Antisera. 

1.  The  "bacterial  antisera  are  produced 
by  injecting-  rabbits  by  intraperi- 
toneally  or  intravenously  with 
emulsions  of  organisms  (either 
broth  cultures  or  salt  solution 
emulsions  of  agar  cultures)  in 
gradually  in^jreasing  quantities  on 
5  or  6  occasions,  at  intervals  of 
from  5  to  6  days.  In  7  to  12  days 
after  the  last  injection,  the  an- 
imal is  bled  and  a  preliminary 
test  made  as  to  the  precipitating 
value  of  the  serum. 
If  this  is  insufficient,  more  injec- 
tions may  be  made.  In  5  to  12 
days  after  the  last  injection  the 
animal  is  bled  and  the  sera  pre- 
served by  sealing-  in  glass  bulbs 


158  BACTERIOLOGY. 


and  kept  in  the  dark  at  a  low 
temperature.  A  preservative  as 
chloroform  may  be  added. 
The  antisera  should  be  absolutely- 
clear.  If  turbid,  it  may  be  filtered 
through  porcelain  candles, 

2.  The  bacterial  filtrates  for  test  are 

produced  by  growing-  the  organ- 
ism in  broth  composed  of  0.5% 
Liebig's  extract  of  beef,  peptone 
1%,  salts  5%  with  a  reaction 
of  -f  5. 

The  cultures  are  incubated  from  a 
week  to  several  months,  and  then 
filtered  through  porcelain  or 
Berfefeld  candles  until  perfectly 
clear. 

The  extracts  may  also  be  made  by 
emulsifying  agar  cultures  in  salt 
solution  and  incubating  them  at 
37°  C.  for  a  week  or  more,  then 
filtering. 

When  the  two  reagents  have  been 
completed,  the  test  is  made  as 
follows: — 

Mix  in  a  series  of  narrow  test 
tubes. 

(a)  Tube  No.  1.  —  0.5  cc.  antibac- 
terial serum  and  1  cc.  bacterial 
filtrate. 

Tube  No.  2  —  0.5  cc.  normal 
serum  and  1  cc.  bacterial  filtrate. 
Tube  No.  3.  —  0.5  cc.  anti  bac- 
teria serum  and  1  cc.  salt  solu- 
tion. 

Tube  No.  4.  —  0.5  cc.  salt  solu- 
tion serum  and  1  cc.  bacterial  fil- 
trate. 

(b)  Place  tubes  in  incubator  at  37*  C. 

(c)  If  test  is  positive,  tube  No.  1 
shows  a  haziness,  which  develops 
Into  a  distinct  cloudiness  or  even 
a  flocculent  ppt.  within  one  hour. 
Tubes  2,  3,  4  remain  clear. 

The  precipitating  antisera  against 
proteid  solutions  are  prepared  by 
methods  analogous  to  those  em- 
ployed for  the  production  of  anti- 
bacterial sera.  If  tests  are  to  be 
made  upon  proteid  material,  as 
(blood  stains,  meat  (as  detection 
of  horse  meat  substitution  for 
beef),  etc.,  they  should  be  ex- 
tracted with  salt  solution,  in  an 
approximate  dilution  of  1-50Q. 


BACTERIOLOGY. 


159 


The  solutions  are  filtered  to  insure 
clearness. 

To  test  the  unknown  proteid  with 
serum  of  an  animal  immunized 
with  the  proteid  sought,  mix  in 
a  series  of  narrow  test  tubes: — 

Tube  No.  1.  —  0.1  cc.  immune  serum 
and  2  cc.  unknown  proteid  solu- 
tion. 

Tube  No.  2.  —  0.1  cc.  immune  serum 
and  2  cc.  known  proteid  solution 
of  variety  suspected  (similarly 
diluted). 

Tube  No.  3.  —  0.1  cc.  Immune  serum 
and  2  cc.  proteid  solution  of  dif- 
ferent nature  (similarly  diluted). 

Tube  No.  4.  —  0.1  cc.  immune  serum 
and  2  cc.  salt  solution. 

Tube  No.  5.  —  2  cc.  unknown  pro- 
teid solution. 

Test  is  positive  when  a  precipitate 
appears  in  tubes  No.  1  and  No.  2, 
but  not  in  any  of  the  others. 


ANTITOXINS. 
Semm  Vaccine  Antltozln. 

Antitoxins  are  produced  for  all  bacteria 
producing-  soluble  toxin,  and  for  the 
toxic  substances  of  a  large  number  of 
other  plant  and  animal  cells.  They 
are  called  antitoxins  because  they 
combine  with  and  render  inert  the 
soluble  toxins.  (See  side-chain  theory). 
They  are  labile  chemical  substances 
which  resist  analysis  or  probably  sim- 
ilar to  euglobulins,  and  are  composed 
of  molecules  of  large  size.  It  was  at 
one  time  supposed  that  antitoxin  was 
but  a  toxin  in  a  different  form.  This, 
of  course,  has  been  disproved.  The 
amount  of  antitoxin  produced  is  much 
greater  than  the  amount  of  toxin 
which  is  injected  or  produced  during 
an  infection.  The  union  between  a 
toxin  and  an  antitoxin  is  of  a  chem- 
ical nature.  The  union  of  these  two 
substances  forms  a  compound  that  is 
harmless  and  differs  from  the  toxin 
and  the  antitoxin  in  that  it  is  much 
more  stable.  Toxins  have  a  greater 
affinity  for  the  three  haptophile  re- 
ceptors of  cells  (free  antitoxin)  than 
for  those  still  associated  with  the 
cells.    The  toxin  and  antitoxin  always 


160  BACTERIOLOGY. 


combine,  if  possible,  before  the  toxin 
and  the  body  cells  enter  into  chemical 
union.  In  certain  cases  when  the 
toxin  has  been  bound  by  the  body 
cells  and  the  antitoxin  is  produced  in 
sufficient  amount  or  injected,  the  toxin 
cell  chemical  union  will  be  broken  up 
and  the  toxin  and  antitoxin  will  com- 
bine. This  is  illustrated  in  diphtheria, 
and  antitoxins  of  this  kind  effect 
cures  because  the  union  between  the 
toxin  and  the  cell  is  comparatively 
unstable.  This  is  not  true  in  cases 
such  as  tetanus,  in  which  the  toxin 
is  so  strongly  combined  with  the  cells 
of  the  nervous  system  and  other  body 
cells  that  it  is  with  difficulty  that 
their  union  is  broken  by  the  addition 
of  antitoxin.  The  union  here  between 
the  toxin  and  the  body  cells  is  so 
stable  that  exceedingly  large  doses  of 
the  antitoxin  are  required,  and  these 
rarely  act  with  any  degree  of  success. 

This  explains  why  tetanus  antitoxin  is 
of  so  little  use  therapeutically.  It  is, 
however,  of  great  use  as  a  prophy- 
lactic when  the  toxin  is  free  and  be- 
ing produced  in  the  body. 

Bhrlich,  in  an  accurate  stud>  of  the 
neutralization  of  the  toxin  by  the 
antitoxin,  noted  that  the  addition  of 
fractional  amounts  of  the  antitoxin 
to  the  Lj°  of  the  toxin  (complete  neu- 
tralization of  one  antitoxic  unit)  and 
the  injection  of  the  resulting  mix- 
tures into  guinea  pigs,  there  was  not 
a  corresponding  decrease  in  the  de- 
gree of  toxicity.  The  toxin,  there- 
fore, seems  to  be  made  up  of  various 
parts;  a  part  seeming  to  have  great 
affinity  for  the  antitoxins  is  not  real- 
h  ly  toxin  and  is  called  Protoxoids. 
These  compose  about  %  of  the  amount 
of  toxins  necessary  to  saturate  one 
immunity  unit.  After  ^  of  the  anti- 
toxin is  added,  the  mixture  of  anti- 
toxin becomes  less  toxic  for  the  ex- 
perimental animals,  down  to  the  point 
where  %  of  the  amount  of  toxin 
necessary  to  saturate  one  unit  of  anti- 
toxin is  used.  This  fraction  is  con- 
sidered the  true  toxin.  Here,  again, 
in  as  much  as  the  toxicity  of  the  mix- 
ture does  not  decline,  it  has  been 
demonstrated  that  it  is  due  to  another 
part  of  the  toxic  molecule  which  has 


BACTERIOLOGY. 


161 


less  avidity  for  the  antitoxin  than  the 
toxin  itself  and  the  protoxoid.  This 
part  of  the  molecule  is  called  epi- 
toxoid,  true  toxoid  or  toxon.  The 
toxon  molecule  necessary  to  saturate 
one  unit  of  antitoxin  is,  therefore, 
made  up  of  1^4  protoxoid,  i/4  true  toxin 
and   %   epitoxoid,  toxoid  or  toxon. 


ANTITOXINS. 

Antitoxins  are  employed  in  the  form 
of  sera  which  may  be  either  liquid, 
dry  or  specificated.  The  immunity  that 
is  produced^  by  the  use  of  antitoxins 
is  passive  and  lasts  for  a  period  of 
a  few  weeks  only.  The  reason  for 
the  Passive  Immunity  is  due  to  the 
fact  that  the  individual  receives  no 
substances  which  stimulate  the  pro- 
duction of  protected  bodies,  but  the 
individual  receives,  however,  those 
protective  antibodies  which  have  been 
produced  in  the  blood  of  some  other 
species.  When  antitoxin  is  injected 
and  becomes  absorbed,  the  neutraliza- 
tion of  these  specific  toxins  takes 
place;  therefore,  it  may  be  used  both 
as  a  prophylactic  and  therapeutic 
agent.  . 

The  most  important  antitoxins  used  at 
the  present  time  are  those  of  diph- 
theria and  tetanus.  Some  other  anti- 
sera  have  been  extensively  used  with 
good  results,  while  others  are  still  in 
the  experimental  stage;  i.  e.,  anti 
streptococci  serum,  anti  dysentery 
serum,  anti  hog  cholera  serum,  anti 
pneumococci  serum  and  anti  tubercle 
serum. 

Biplitherltic  antitoxin. 

The  organism  is  grown  on  Loeflfler's 
blood  serum  in  the  incubator  at  37°  C, 
care  being  taken  that  the  culture  is 
pure. 

The  pure  diphtheria  culture  is  now 
transferred  to  large  flasks  of  beef 
broth  and  incubated  at  37°  C.  for  a 
period  of  about  two  weeks,  during 
which  time,  the  rapid  growth  of  the 
organism  has  elaborated  its  specific 
toxin  and  thrown  it  off  into  the  broth. 
The  culture  is  now  examined  micro- 
scopically in  order  to  determine  the 
absence  of  contamination.  A  pre- 
servative,   such   as   carbolic   acid  or 


162 


BACTERIOLOGY. 


trikresol,  is  added  and  the  culture 
then  passed  through  a  Burkef  eld  filter. 
The  diphtheritic  toxin  (filtrate)  Is 
then  placed  in  the  refrigerator  until 
wanted  for  use. 

The  horses  used  in  the  manufacture  ot 
anti  diphtheritic  serum  are  removed 
from  the  detention  stable  where  they 
have  been  confined  for  several  weeks, 
during  which  time  they  are  subjected 
to  a  thorough  physical  examination 
and  tested  for  the  presence  of  gland- 
ers by  the  Mallein  test.  They  are  now 
admitted  to  the  antitoxin  stable  and 
injected  subcutaneously  with  the 
diphtheria  toxin.  The  first  dose  in- 
jected is  but  a  fraction  of  a  cc,  then 
increasingly  larger  doses  are  injected 
until  the  animal  is  able  to  receive 
300  cc.  or  more  at  a  single  injection. 
The  intervals  between  the  injections 
and  the  rate  of  increase  in  the  doses 
at  any  time  depends  upon  the  condi- 
tion of  the  animal.  In  order  that  a 
constant  process  of  antitoxin  forma- 
tion may  take  place  in  the  body  of 
the  horse,  and  that  a  potent  serum 
may  be  produced,  the  Injection  of  the 

»  toxin  should  be  made  as  rapidly  as 
the  reactions,  which  follow  each  in- 
jection, will  allow. 

The  toxin  treatment  usually  occupies  a 
period  of  about  six  weeks,  after  which 
the  horse  is  allowed  to  rest  for  about 
two  weeks  in  order  that  all  of  the 
toxin  injected  may  be  absorbed. 

By  means  of  a  sterile  canula,  rubber 
tube  and  glass  cylinders,  the  animal 
is  now  bled  from  the  jugular  vein 
under  the  proper  aseptic  and  anti- 
septic conditions  by  securing  as  much 
blood  as  the  horse  can  conveniently 
yield.  After  the  serum  separates, 
usually  at  the  end  of  24  to  48  hours, 
the  clear  fiuid  is  poured  into  large, 
serile,  glass  containers.  A  preserva- 
tive is  added  and  the  material  is 
transferred  to  the  laboratory,  where 
it  is  filtered  through  a  Burkefeld 
filter. 

The  serum  is  now  submitted  to  tests  as 
to  potency,  safety  and  microbial  con- 
tamination. 

Potency  Test.  Varying  amounts  of 
serum  are  mixed  with  the  L  +  dose 
of  diphtheria  toxin  ancl  injected  into 


BACTERIOLOGY. 


163 


a  series  of  guinea  pigs,  each  weigh- 
ing 250  gms. 

(The  L  +  dose  of  toxin  is  the  least 
amount  of  toxin  which,  when  mixed 
with  one  unit  of  standard  antitoxin 
and  injected  into  a  guinea  pig  of  250 
gm.  weight,  is  sufficient  to  kill  the 
animal  in  four  days. 

By  this  test  it  is  possible  to  determine 
the  smallest  amount  of  antitoxin 
which  will  protect  the  guinea  pig  of 
250  gm.  weight  when  the  animal  has 
received  simultaneously  the  L  -f  dose 
of  toxin.  This  minimum  amount  of 
antitoxin  represents  one  unit.  If  one 
five  hundredth  cc.  of  the  antitoxin 
represents  the  smallest  amount  which 
is  capable  of  neutralizing  the  L  + 
dose  of  toxin,  the  antitoxin  then  pos- 
sesses a  potency  of  500  units  per  cc.) 

Safety  Test.  Several  guinea  pigs  are 
each  injected  subcutaneously  with 
2  cc.  of  the  serum  and  held  under 
close  observation  until  satisfied  that 
the  serum  contains  no  injurious 
properties. 

Microbial  contamination.  Inoculate  cul- 
ture media  with  large  amounts  of 
antitoxin  under  aerobic  and  anaerobic 
conditions.  Place  in  the  incubator, 
and  if  after  a  period  of  72  hours  no 
growth  occurs,  the  serum  is  read^  for 
use.  If,  however,  a  growth  appears, 
the  serum  is  refiltered  and  re- 
examined for  microbial  contamination. 

The  serum  is  now  put  up  in  sterile  glass 
cylinders  so  constructed  that  steril- 
ized needles  and  pistons  may  be  ap- 
plied and  the  antitoxin  injected  di- 
rectly from  the  containers.  Each 
container  bears  a  label  indicating  the 
number  of  antitoxin  units  enclosed 
and  the  date  of  preparation.  A  num- 
ber of  these  packages  are  now  opened 
and  examined  for  contamination,  and, 
if  free  from  this,  the  serum  is  ready 
for  distribution. 

Tetanus  Antitoxin. 

The  preparation  of  Tetanus  Antitoxin 
differs  but  little  from  that  of  the 
diphtheritic  antitoxin.  A  pure  culture 
of  bacillus  tetani  is  inoculated  into 
large  flasks  of  glucose  bouillon  and 
placed  under  anaerobic  conditions 
•  (see  anaerobic  cultivations),  or,  be^ 
fore   inoculation,   drive   oft   the  free 


164  BACTERIOLOGY. 


oxyg-en  by  boiling^  the  glucose  bouil- 
lon and  then  covering  the  liquid  me- 
dium by  a  layer  of  oil.  Incubate 
these  cultures  at  37°  C.  for  several 
weeks,  after  which  examine  micro-, 
scopically;  add  a  preservative  and* 
pass  the  culture  through  a  Burkefeld 
filter  and  then  through  Pasteur  filter. 

The  filtraton  process  had  better  be  car- 
ried out  in  an  isolated  room  used  only 
for  the  preparation  of  tetanus  toxin 
on  account  of  the  danger  of  contam- 
inating any  other  material  or  biolog- 
ical products  with  the  tetanus  bacillus^^ 

The  tetanus  antitoxin  is  obtained  by 
injecting  horses  with  the  toxin  along 
the  lines  laid  down  in  the  preparation 
of  diphtheritic  antitoxin.  The  serum 
is  tested  relative  to  potency,  safety 
and  absence  from  microbial  contam- 
ination. 

The  unit  of  tentanus  antitoxin  is  ten 
times  the  least  quantity  of  antitetanic 
serum  necessary  to  save  the  life  of  a 
300  gm.  guinea  pig  for  96  hours, 
against  the  official  dose  of  a  standard 
toxin  furnished  by  the  hygienic  lab- 
oratory of .  the  Public  Health  and 
Marine  Hospital  Service. 

Anti  Streptococci  Serum.  Bouillon  cul- 
tures of  the  streptococcus  pyogenes 
ar??  killed  by  heating  and  injected  into 
horses  in  increasingly  larger  doses. 
Generally  but  one  strain  of  the  or- 
ganism is  used  and  the  serum  is 
called  "Monovalent."  Frequently,  how- 
ever, several  strains  of  the  organism 
are  used  and  the  serum  is  then  desig- 
nated "Polyvalent."  The  procuring 
of  the  serum,  etc.,  is  carried  out  along 
the  lines  laid  down  in  the  preparation 
of  the  anti  diphtheritic  serum.  The 
obtained  antitoxin  is  tested  in  regard 
to  safety  and  freedom  from  microbial 
contamination,  but  not  as  to  potency, 
in  as  much  as  there  are  no  known 
methods  of  standardizing  the  product. 

Anti  Gonococcus  Serum.  Killed  cultures 
of  M.  Gonorrhoea  are  injected  in- 
traperitoneally  into  large,  healthy 
rams  in  increasingly  larger  doses; 
finally  live  cultures  are  injected.  The 
degree  of  acquired  immunity  is  de- 
termined by  frequent  agglutination 
tests.  The  serum  is  tested  as  to 
safety   and   freedom   from  microbial 


BACTERIOLOGY. 


165 


contamination  but  not  as  to  potency. 
Anti  Dysentery  Serum.  Both  Monova- 
lent and  Polyvalent  antitoxic  sera  for 
epidemic  dysentery  have  been  pre- 
pared b5^  Shig-a  by  injecting  horses 
with  the  filtrate  from  bouillon  cul- 
tures of  the  bacillus.  It  is  still  in 
the  experimental  stage. 

VACCINES. 

Preventative  medicine  depends  to  a  con- 
siderable extent  upon  the  use  of  vac- 
cines, antitoxins  and  certain  other 
specific  biological  preparations,  such  as 
diphtheritic  antitoxin,  small-pox  vac- 
cines, tuberculins,  etc.  As  stated  in 
other  parts  of  the  book,  the  infection 
of  the  animal  organism  is  due  to  the 
absence  of  natural  or  acquired  re- 
sistance. 

An  acquired  resistance  or  immunity 
may,  therefore,  be  brought  about  by 
the  application  of  a  vaccine  or  an 
antitoxin.  The  application  of  small- 
pox vaccine  (although  believed  not 
to  be  bacterial  in  origin  but  will 
illustrate  the  point  in  question) 
causes  a  reaction  in  the  body,  or  a 
mild  form  of  the  disease,  and  brings 
about  an  active  immunity  which  is 
relatively  permanent  in  duration.  The 
use  of  diphtheritic  antitoxins  causes 
a  passive  immunity  and  affords  temp- 
orary protection  by  neutralizing-  the 
diphtheritic  toxin  molecules. 

Vaccines  are  weakened  or  modified  ^ 
viruses.  Small-pox,  black-leg-  and 
anthrax  vaccines  may  be  used  with 
safety  only  on  individuals  free  from 
the  specific  disease  in  question,  be- 
cause if  given  to  an  individual  suf- 
fering from  the  specific  disease,  the 
introduction  of  the  attenuated  or- 
ganisms or  virus,  would  tend  to  in- 
crease the  infection.  The  g-eneral  ac- 
tion of  these  vaccines  is,  therefore, 
preventative,  or  prophylactic,  and  not 
curative. 

There  are  several  methods  employed  in 
the  preparation  of  vaccines.  The  gen- 
eral plan  is  to  attenuate  or  modify 
the  viruses  so  that  they  may  be  in- 
jected into  the  normal  animal  body 
without  danger  of  producing  serious 
diseased  lesions.  (See  active  immuni- 
zation). 


166  BACTERIOLOGY. 


The    following    methods    are  usually 
used: — 

(1)  Attenuation  by  growth  at  a  temp- 
erature above  the  optimum  (see 
anthrax  vaccine). 

(2)  Attenuation  by  passage  of  virus 
through  some  species  other  than 
the  animals  for  which  the  virus 
is  specific  (see  small-pox  vac- 
cine). 

<3)  Attenuation  of  virus  by  drying 
at  constant  temperature  (see 
Rabbi's  treatment). 

(4)  Attenuation  by  chemical.  Patho- 
genic bacteria  are  grown  in  the 
presence  of  weak  antiseptics, 
which  weakens  their  disease-pro- 
ducing powers. 

(5)  By  the  simultaneous  injection  of 
a  virus  together  with  its  pro- 
tective serum  (hog  cholera). 

(6)  By  the  combination  of  pathogenic 
bacteria  with  bacteria  of  other 
species  antagonistic  to  them,  as 
illustrated  by  the  restraining  ac- 
tion of  yeast  upon  pyogenic  bac- 
teria and  antagonism  of  the  Ps. 
pyocyanea  toward  the  bacterium 
anthracis. 

.  (7)  The  filtration  of  liquid  cultures 
of  pathogenic  organism  and  the 
separation  of  the  organism  from 
the  toxin  (the  toxin  is  used  to 
immunize  animals  in  the  produc- 
tion of  antitoxin). 
(8)  The  liestruction  of  young  living 
cultures  of  specific  bacteria  by 
moist  heat  at  a  temperature 
slightly  above  their  thermol  death 
point. 

Anthrax  Vaccine.     (Pasteur's  Method). 

Cultivate    the    bacterium    from  the 

blood  of  a  typical  case  of  anthrax  or 

agar  broth. 
Prepare  two  vaccines  as  follows: — 

Vaccine  No.  1.  (less  active).  Grow 
the  anthrax  organisms  at  a  temper- 
ature of  42°  C.  for  a  period  of  15 
to  20  days  (this  produces  an  as- 
porogenous  race).  At  the  end  of 
this  time,  suspend  cultures  in  a 
sterile   physiological   salt  solution. 

Vaccine  No.  2.  Same  as  No.  1,  except 
that  it  is  grown  for  10  to  15  days. 
Both  vaccines  must  now  be  tested 
for  activity  and  safety  by  animal 


BACTERIOLOGY.  167 


inoculation.  No.  1  should  kill  white 
mice,  but  should  not  cause  death  in 
guinea  pigs  or  rabbits.  No.  2 
should  kill  white  mice  and  guinea 
pigs  but  not  rabbits. 
Healthy  animals  are  injected  sub- 
cutaneously  with  1  cc.  of  No.  1. 
From  several  days  to  a  few  weeks 
after  the  injection  of  No.  1,  the 
second  vaccine  is  injected.  A  severe 
reaction  with  occasional  death  fol- 
lows the  use  of  the  vaccine.  These 
accidents  can  be  attributed  to  care- 
less methods  in  standardizing  and 
administering  the  vaccine.  The  ob- 
jection to  this  method  lies  in  the 
danger  of  using  a  living  organism. 
Good  results  have  been  obtained 
from  the  use  of  killed  and  dried 
anthrax  organisms. 

Small-pox  Vaccine.     The  first  method 
employed    in    small-pox  vaccinations 
was    inoculating   healthy  individuals 
with  the  virus  from  a  mild  case  of 
'    the    disease.      Since    1796  small-pox 
vaccination  has  been  carried  out  by 
vaccinating  with  small-pox  virus.  As 
yet,  it  has  not  been  conclusively  de- 
termined that  the  cow-pox  of  cattle 
and  the  small-pox  of  man  possess  in- 
timately related  causative  factors,  but 
abundant  evidence  proves  the  efficacy 
of  cow-pox  virus  as  a  specific  prophy- 
lactic against  small-pox  in  man. 
Under  sterile  conditions,  the  virus,  or 
seed,  is  secured  by  removing  the  ex- 
tradite from  the  vesicles  on  -infected 
heifers.    This  virus  is  now  inoculated 
into   calves   or   yearlings    that  have 
been  placed  in  detention  stables  where 
they    are     inspected    and  carefully 
tested  for  tuberculosis.    Before  their 
admittance  to  the  vaccine  laboratory, 
they  have  passed  as  healthy  in  every 
way     and    have    had     their  bodies 
scrubbed  with  soap  and  water  and  a 
weak  antiseptic  solution. 
The  virus  is  inoculated  as  follows: — 
The  ventral  surface  of  the  body  is 
shaved    and    cleansed,    and  under 
sterile  conditions,  the  skin  is  scari- 
fied in  parallel  lines  over  the  greater 
portion  of  the  abdomen.    The  stock 
virus   is   inoculated   clear  through 
the  scarified  area.     The  animal  is 
then  placed  in  the  propagating  room 


168  BACTERIOLOGY. 


and  all  possible  precautions  taken 
to  avoid  contamination  by  bacteria. 
In  from  5  to  7  days,  characteristic 
vesicles  appear  on  the  inoculated 
area,  filled  with  a  thick,  heavy  ex- 
tradite. Animal  removed  to  the 
operating-  table,  the  field  is  washed 
with  sterile  water  and  the  contents 
of  the  vesicles  are  removed  with 
^  a  sterile  pipette.  (By  recent  order 
of  the  Federal  Government,  animals 
used  in  this  work  must  be  slaught- 
ered before  the  vaccine  is  obtained 
and  then  carefully  autopsyed).  The 
cow-pox  extradite  is  mixed  with 
about  50%  glycerine,  which  adds  as 
a  preservative.  Safety  tests  are 
made  by  inoculation  of  small  por- 
tions into  guinea  pigs.  It  is  then 
placed  in  the  refrigerator.  The 
glycerine  and  low  temperature 
gradually  destroy  extraneous  con- 
tamination. Potency  tests  are  made 
on  calves  and  rabbits  or  guinea 
pigs.  Inoculations  are  made  in  the  • 
culture  media  to  exclude  both 
aerobic  and  anaerobic  bacteria. 
For  the  presence  of  the  tetanus  ba- 
cillus 1  cc.  of  the  product  is  trans- 
ferred to  glucose  beef  bouillon  and 
placed  under  anaerobic  conditions  at 
a  temperature  of  3  71/^*"  C.  for  about 
10  days.  Any  growth  is  removed 
by  filtration  and  the  filtrate  is  in- 
jected in  the  guinea  pig.  Absence 
of  symptoms  in  the  injected  animals 
shows  the  absence  of  tetanus  toxin 
in  the  cultures.  At  the  completion 
of  these  tests,  the  product  is  placed 
in  small  capillary  tubes  or  upon 
ivory  points,  sealed  in  glass  con- 
tainers. 

If  kept  in  a  cool,  dark  place,  it  will 
retain  its  activity  for  a  period  of 
about  8  months. 

Babies  Vaccine.  Originated  by  Pasteur 
in  1885,  with  slight  modifications  it 
continues  to  be  the  only  specific  treat- 
ment for  rabies. 

Method.  A  dog  suffering  from  rabies  is  . 
killed,  a  small  portion  of  the  brain 
removed,  emulsified  in  sterile  water 
or  salt  solution;  inject  a  few  drops 
of  the  emulsion  subdurally  into  a 
rabbit.  This  inoculation  should  pro- 
duce symptoms  of  "dumb  rabies"  and 


BACTERIOLOGY. 


169 


causer  the  death  of  the  rabbit  in  14 
to  18  days.  The  virulence  of  the 
strain  of  rabbit  material  is  increased 
by  making-  subdural  inoculations  until 
the  incubation  is  shortened  to  about 
six  days.  When  the  rabbit  shows 
symptoms  on  the  sixth  or  seventh  day 
after  the  inoculation,  the  virulence  of 
the  virus  is  called  fixed  virus  and  is 
now  used  for  the  preparation  of  the 
vaccine. 

The  spinal  cord  of  the  rabbit  dying 
within  seven  days  is  removed  anti- 
septically  and  suspended  over  caustic 
potash  and  dried  at  a  temperature  of 
23°  C.  for  a  period  of  from  1  to  15 
days.  The  patient  is  now  vaccinated 
with  a  suspension  of  a  spinal  cord 
which  has  been  attenuated  by  drying" 
for  14  or  15  days.  On  the  succeeding 
days  of  the  treatment,  the  injection  is 
made  of  spinal  cord  which  has  been 
less  and  less  attenuated. 

Treatment  usually  lasts  about  21  days, 
or  until  the  patient  has  received  an 
injection  of  the  least  attenuated  virus. 
It  is  very  important  that  the  treat- 
ment be  begun  as  early  as  possible, 
ment  be  begun  as  early  as  possible, 
when  bitten  by  a  rabid  animal,  in 
secured  before  the  expiration  of  the 
incubation  period. 

Hog"  Cholera  Vaccine.  (Dorset-Niles 
Serum).  Obtain  the  hog  cholera  virus 
by  withdrawing  the  blood  from  the 
carotid  artery  suffering  from  the  dis- 
ease. Test  it  for  activity,  as  a  given 
strain  of  virus  may  not  produce  the 
acute  form  of  hog  cholera.  Raise  the 
virulence,  if  necessary,  by  passing 
through  a  series  of  young  pigs  until  it 
uniformly  produces  symptoms  in  4 
to  6  days  and  death  is  less  than  15 
days.  This  degree  of  virulence  is 
necessary  in  manufacturing  the  serum. 

The  blood  used  in  the  process  of  hyper- 
immunization  should  be  obtained  from 
susceptible  pigs,  weighing  from  50  to 
100  pounds  each.  The  animals  used 
as  hyperimmunes  should  be  healthy 
and  weigh  from  100  to  300  pounds 
and  possess  either  natural  or  acquired 
immunity  to  the  disease.  The  blood  is 
secured  from  the  diseased  pig  by  al- 
lowing the  blood  to  flow  from  the 
jugular  vein  into  a  sterile  pan,  or  by 


170  BACTERIOLOGY. 


drawing:  it  under  aseptic  conditions 
from  the  carotid  artery.  The  blood 
obtained  is  defibrinated  and  the  serum 
is  secured. 

The  immune  hog"s  are  hyperimmunized 
by  the  slow  or  by  the  quick  method. 

The  slow  method.  The  animals  receive 
several  injections  at  intervals  of 
every  few  days,  each  succeeding  dose 
of  the  virus  being  increased  in  pro- 
portion to  the  weight  of  the  animal. 

The  quick  method.  Animal  is  injected 
with  one  large  dose  of  the  virus,  the 
amount  determined  by  the  weight  of 
the  aninlal. 

In  from  one  to  two  weeks  after  the 
hyperimmune  animal  has  received  the 
last  injection  of  the  virus,  the  end  of 
the  tail  is  severed  with  a  sharp  in- 
strument and  several  hundred  cc.  of 
blood  collected  defibrinated,  a  pre- 
servative added  and  placed  in  the  re- 
frigerator. Repeat  this  process  sev- 
eral times  at  7  to  10  day  intervals, 
when  the  animal  is  ready  for  rehyper- 
immunizing. 

In  rehyperimmunizing  the  animal  about 
y2  of  the  quantity  of  the  virus  used 
in  the  first  process  is  injected.  Re- 

*  hyperimmunize  2  or  3  times,  then 
relieve  the  animal  of  all  its  blood. 
Mix  the  different  lots  of  serum  repre- 
senting the  different  bleedings  and 
test  the  potency  of  the  product  by 
injecting  subcutaneously  four  sus- 
ceptible pigs  weighing  about  50 
pounds  with  2  cc.  of  the  virus.  Two 
of  these  pigs  are  simultaneously  in- 
jected with  about  20  cc.  of  the  serum. 
If  the  serum  possesses  the  necessary 
activity,  the  two  test  pigs  will  re- 
main normal,  except  for  a  thermol 
reaction  and  slight*  clinical  symp- 
toms, while  the  two  controlled  pigs 
should  show  symptoms  in  5  or  6  days 
and  die  in  less  than  15  days. 

The  treatment  now  consists  in  simul- 
taneously injecting  hyperimmune  se- 
rum and  virus  intramuscularly  into 
healthy  hogs.  The  amount  of  hyper- 
immune serum  injected  varies  from 
10  to  70  cc,  depending  upon  the 
weight  of  the  hog. 

Other  Vaccines  in  General. 

After  Pasteur  had  shown  that  It  was 
possible  to  produce  active  immunity 


BACTERIOLOGY.  171 

in  animals  by  substituting:  chicken 
cholera,  anthrax  and  swine  plague,  the 
thought  naturally  suggested  itself 
that  the  same  should  be  possible  in 
the  case  of  some  of  the  organisms 
which  are  pathogenic  to  man.  At- 
tempts in  this  direction  showed  that 
it  was  not  only  impossible  to  protect 
laboratory  animals  against  infections 
like  typhoid  fever  and  cholera,  but 
man  could  also  be  protected,  not  only 
by  the  use  of  living  cultures,  but 
even  with  the  killed  organisms.  The; 
great  question  naturally  has  been  how 
large  a  dose  of  bacterial  should  be 
injected  and  how  frequently  the  in- 
jection should  be  made  in  order  that 
one  might  secure  sufficient  protection. 
Pfeiffer  and  Kolle  were  the  first  to 
attempt  this  in  a  human  being  through 
a  bacterialytic  content  of  the  serum. 
Wright  then  introduced  a  method  in 
which  he  thought  that  by  the  opsonic 
contents  of  the  blood  the  degree  of 
protection  might  be  indicated.  It  has 
been  shown,  however,  that  a  parallel 
between  the  size  of  the  dose  and  the 
serum  content  of  protective  sub- 
stances and  the  degree  of  immunity 
does  not  exist,  and  the  method  now 
employed  are  the  outcome  of  actual 
triumph. 

Freparation  of  Vaccines. 

Typhoid  Pever.  The  culture  from 
which  the  vaccine  is  to  be  made  is 
brought  to  a  certain  degree  of  viru- 
lence by  passage  through  animals 
and  that  when  grown  in  bouillon  it 
should  yield  from  one  thousand  to 
two  thousand  million  bacilli  per  cc. 
(This  procedure  is  not  absolutely 
necessary).  The  vaccine  should,, 
however,  be  polyvalent;  i.  e.,  it 
should  be  made  from  a  number  or 
different  strains.  The  medium  for 
the  growth  is  generally  a  1%  pep- 
tone broth.  Each  of  the  strains  is 
inoculated  into  separate  fiasks  and 
grown  at  37  C.  for  24  to  48  hours, 
after  which  they  are  carefully 
mixed  and  sterilized  in  a  water 
bath  at  60°  C.  (Some  authorities 
advocate  52*'  C.)  The  flask  is  to 
remain  in  the  hot  water  for  10  to 
15  minutes  and  then  removed. 
(Some  authorities   indicate   an  ex- 


172  BACTERIOLOGY. 


posure  for  1  hour.  Necessity  of  this 
questioned).  The  sterility  of  the 
contents  is  now  tested  by  placing 
.1  to  10  cc,  according  to  the  amount 
of  the  material,  in  agar  or  liy  in- 
oculating broth. 

Determine  the  niimber  of  bacteria  per 
cc.  by  Wright's  method.  Mark  a 
capillary  pipette  v/ith  a  glass  pencil 
about  %  of  an  inch  from  the  end, 
puncture  the  thumb  and  charge  the 
pipette  with  a  volume  of  blood  as 
indicated  by  the  mark  on  the  pi- 
pette. Now  charge  the  pipette  with 
a  like  volume  of  the  bacterial  emul- 
sion and  then  with  three  volumes 
of  a  9/10%  salt  solution  (keeping 
the  individual  portions  separate 
from  one  another  by  little  air  bub- 
bles). The  blood  and  bacterid  are 
now  thoroughly  mixed  by  repeated- 
ly blowing  the  contents  of  the  capil- 
lary pipette  upon  the  slide.  Small 
drops  of  this  emulsion  are  now 
mounted  on  a  clean  slide  and  spread 
out  like  a  blood  film,  dried  and 
stained  with  Jenner's  or  Hastiiig's 
stain.  A  small  square  diaphragm 
of  paper  is  placed  in  the  ocular  of 
the  microscope,  the  "red  cells  and 
bacteria  are  counted  in  successive 
filed  until  1000  of, the  red  cells  have 
been  counted.  The  number  of  red 
cells  in  one  cc.  of  blood  is  ap- 
proximately 5,000,000,  and  as  the 
red  cells  a.nd  the  bacteria  must  be 
present  in  the  same  ratio  to  one 
another  as  in  the  original  units  of- 
volume,  the  number  of  bacteria  per 
cc.  of  the  vaccine  is  ascertained  ac- 
cording to  equation:  Number  of 
red  cells  counted  :  number  of  bac- 
bacteria  counted   :    :   5,000,000   :  X. 

A  more  accurate  method  has  been 
suggested  by  Hopkin  based  upon  the 
concentration  of  bacterial  culture 
by  centrifugation  and  the  prepara- 
tion of  standard  emulsions  from  the 
sediment.  This  requires  an  espe- 
cially constructed  centrifugation 
tube,  which  is  prepared  by  the 
International  Instrument  Company, 
Cambridge,  Mass. 

Wright  recommends  a  first  injection 
of  750.000.000  to  1,000.000,000  organ- 


BACTERIOLOGY. 


173 


isms  and  double  this  amount  for  the 
second  injection. 
Fearing  that  the  injection  of  a  large 
dose  of  organisms  may  be  followed 
by  a  diminution  in  the  protective 
substances  of  the  body  (negative 
phase),  owing  to  an  interaction  be- 
tween the  normal  antibacterial  sub' 
stances  and  the  bacterial  antigen, 
the  individual  may  be  temporarily 
less  resistant  to  the  corresponding 
infection.  Wright,  therefore,  sug- 
gests that  in  persons  who  are  likely 
^to  be  exposed  to  typhoid  fever  soon 
^  after  the  first  injection,  this  should 
be  smaller  than  usual,  and  that  its 
effect  is  to  be  supplemented  later 
by  a  correspondingly  stronger  injec- 
tion. 

Anti-typhoid  vaccination,  as  indicated 
above,  is  for  prophylaxis  only. 
Various  attempts,  however,  have 
been  made  to  use  it  as  a  curative 
agent.  Some  writers  have  expressed 
themselves  favorably  upon  this 
point;  others  condemn  it  strongly. 
At  any  rate,  it  will  require  a  great 
deal  of  investigation  before  definite 
conclusions  can  be  reached.  It  is 
impossible  to  tell  when  and  how 
much  we  inject,  or  as  to  whether 
it  is  beneficial  or  harmless,  unless 
Wright's  opsonic  index  will  prove 
of  some  value  as  determined  by 
future  study. 
Asiatic  Cholera. 

Two  methods  of  vaccination  against 
this  disease  have  been  carried  out 
with  positive  results  in  both  meth- 
ods. 

Haffkine's  method  depends  upon  the 
use  of  the  cholera  spirillum  after  it 
has  been  attenuated  by  the  growth 
at  temperature  above  the  optimum. 
Vaccines  of  different  strengths  are  ' 
used. 

Kolle's  method  depends  upon  the  use 
of  heated   (killed)   cultures   of  the 
organisms. 
Bubonic  PlagTie. 

The  same  methods  employed  in  the 
vaccination  against  Asiatic  cholera 
are  used  in  the  vaccination  against 
Bubonic  Plague.  Cultures  of  the , 
plague  bacillus,  killed  by  heating  at 
a  temperature  of  60®  C.  for  1  hour. 


174  BACTERIOLOGY. 


Bacterial    Vaccines    or    Bacterins  for 
Therapeutic  Purposes. 

Recent  studies  would  tend  to  show 
that  bacterins  (killed  bacteria)  may- 
be employed  as  curative  agents  in 
those  infections  which  tend  to 
chronicity,  and  in  which  toxins  play 
little  or  nor  part.  Wright  and 
Doug-las  first  advanced  the  theory 
of  opsonins  including  the  suggestion 
that  the  subcutaneous  injection  of 
a  given  species  of  bacteria,  killed 
by  heating,  conferred  to  the  blood 
when  injected  a  greater  opsonic 
activity  towards  the  species  of  or- 
ganisms  in  question. 

In  preparing  bacterial  vaccines,  (based 
upon  the  opsonic  theory),  the  spe- 
cific organism  is  isolated,  grown  for 
24  hours  at  37°  C,  emulsified  in 
sterile  physiological  salt  solution, 
heated  in  a  water  bath  at  60°  C.  for 
^  hour,  standardized  as  to  number 
of  bacteria  in  1  cc,  and  a  preserva- 
tive added  (making  the  emulsion 
correspond  to  V2%  lysol  and  tested 
for  its  sterility  as  in  preparation 
of  typhoid  bacteria). 

The  use  of  these  bacterial  vaccines 
has  brought  splendid  results  in  the 
treatment  of  furunculosis,  acne, 
sycosis  and  other  infections  caused 
t>y  pyogenic  organisms. 

Two  kinds  of  vaccine  are  used: — the 
so-called  autogenous  vaccines;  i.  e., 
vaccines  that  are  derived  from  the 
individual  organism  which  is  re- 
sponsible for  the  particular  infec- 
tion, and  the  so-called  stock  vac- 
cines, which  are  prepared  from 
stock  cultures  of  the  specific  or- 
ganisms responsible  for  the  infec- 
tion. 

The  question  as  to  whether  or  not 
autogenous  vaccines  are  imperative 
has  created  a  great  deal  of  discus- 
sion. It  would  seem  theoretically, 
at  least,  that  the  probable  existence 
of  many  strains  of  a  given  type  of 
organism  would  make  the  autogen- 
ous vaccines  preferable  to  the  stock 
vaccines. 

TUBERCULINS. 

Koch's  Tuherculln.     (Old).     An  inocu- 
lation of  the  bovine  or  liuman  bact, 


BACTERIOLOGY. 


175 


tuberculosis  is  made  into  several 
flasks  of  beef  bouillon  to  which  5% 
glycerine  has  been  added.  The  cul- 
tures are  carefully,  placed  on  the  sur- 
face of  the  medium.  After  an  incu- 
bation at  37°  to  38°  C.  for  a  period  of 
6  to  10  weeks  or  longer,  the  growth 
that  slowly  spreads  over  the  surface 
finally  falls  to  the  bottom  (it  is  neces- 
sary that  during  the  incubation,  the 
cultures  remain  undisturbed  and  have 
access  to  plenty  of  air  without  temp- 
erature fluctuations  in  order  that  they 
may  complete  the  elaboration  of  ac- 
tive tuberculinic  substance).  The  cul- 
tures are  removed  from  the  incubator 
and  sterilized  in  streaming  steam. 
Evaporate  the  cultures  over  a  water 
bath  to  0.1  its  original  volume;  re- 
move the  bacteria  by  passing  the 
cultures  through  filter  paper  and  a 
Burkefeld  filter;  add  a  preservative. 

The  active  substance  of  a  tuberculin  is 
apparently  an  albuminous  derivative 
insoluble  in  alcohol  and  is  elaborated 
during  the  organism's  multiplication. 
The  product  used  is  harmless  for 
healthy  animals,  but  exerts  a  toxic 
action  upon  those  affected  with  tuber- 
culosis. This  tuberculin  is  used  as  a 
diagnostic  agent,  not  as  a  prophylactic 
agent.  Its  injection  into  individuals 
affected  with  tuberculosis  is  followed 
in  from  2  to  10  hours  by  a  rise  of 
temperature,  which  continues  for  a 
few  hours  then  subsides. 

The  dose  of  this  tuberculin  for  cattle 
is  0.25  cc.  20.7  cc.  By  reason  of  its 
syrupy  consistency  and  small  dose,  it 
is  usually  diluted  with  seven  parts  of 
weak  carbolic  acid  solution;  2  cc.  of 
the  diluted  tuberculin  is  used  as  the 
dose  for  cattle.  The  product  is  tested 
for  activity  by  injecting  known  tuber- 
culosus  animals  and  the  activity  of 
the  product  is  indicated  by  the  typical 
reaction  which  follows. 

Other  methods  than  the  one  described 
above  of  applying  tuberculin  as  a  di- 
agnostic agent  have  been  instituted  by 
Kalmette,  von  Pirquet  and  Morrow. 

Kalmette's  Method  consists  in  the  instil- 
lation in  the  eye  of  one  drop  of  a 
1%  solution  Koch's  purified  or  refined 
tuberculin  (prepared  by  treating  the 
original  tuberculin  r  with  absolute  al- 


176  BACTERIOLOGY. 


cohol,  washing  and  drying  the  pre- 
cipitate). A  positive  reaction  is  in- 
dicated by  a  congestion  of  the  pal- 
pebral and  ocular  conjunctiva  a  few 
hours  after  its  application. 

Von  Pirquet's  Method.  The  patient's 
arm  is  cleansed;  one  drop  of  tubercu- 
lin (old)  is  placed  on  the  skin  of  the 
cleansed  area  and  the  skin  underneath 
the  drop  is  scarified.  Two  or  more 
areas  are  treated  in  this  way.  It  is 
well  to  scarify  another  small  area 
as  a  control,  this  area  to  be  treated 
with  a  drop  of  sterile  salt  solution, 
or  a  solution  of  glycerijae  and  dilute 
carbolic  acid  in  substitution  for  the 
tuberculin. 

The  appearance  of  a  reddish  zone  in 
from  12  to  24  hours  under  the  tuber- 
culin areas  indicates  a  positive  reac- 
tion. 

Morrow's  Method.  An  ointment  is  pre- 
pared from  equal  parts  of  tuberculin 
(old)  and  hydrous  lanolin  and  vigor- 
ously rubbed  on  a  small  portion  of  the 
skin  of  the  abdomen.  A  distinct 
granular  or  papular  eruption  at  the 
point  of  application  after  about  24 
•  hours  indicated  a  positive  reaction. 

Kocli's  Tulberculin  in  "T.  R."  (tuber- 
culin residum)  is  prepared  by  repeat- 
ed centrifugation  of  a  suspension  in 
water  of  the  dried  and  ground  organ- 
isms. The  supernatent  fluid  "t.O." 
after  the  first  centrifugalization  is 
discarded,  and  the  final  product,  con- 
sisting of  the  constituents  of  the  bac- 
teria which  are  insoluble  in  water, 
contains  the  T.  R.  1  cc.  of  the  tuber- 
culin T.  R.  should  contain  the  equiva- 
lent of  1  mg.  of  the  dry  tubercle 
solids. 

Koch's  Tuberculin  "B.  E."  (bacillary 
emulsion)  is  composed  of  a  suspen- 
sion of  crushed  or  thoroughly  ground 
tubercle  bacilli  in  5%  glycerine  solu- 
tion. Each  cc.  should  contain  the 
equivalent  of  1  mg.  of  tubercle  solids. 

Koch's  T.  R.  and  B.  E.  are  used  as 
therapeutic  agents,  the  B.  E.  being 
regarded  most  favorably  b3^  clinicians. 
They  are  administered  by  subcutane- 
ous injections.  The  initial  dose 
recommended  by  Wright  is  one  four 
hundredth  to  six  hundreth  mg.  The 
Intervals  between  the  successive  treat- 


BACTERIOLOGY.  177 


ments  varies  from  three  to  ten  days. 


MALLEIN. 

Mallein  is  used  for  the  diagnosis  of 
glanders.  It  is  prepared  from  cul- 
tures of  the  bact.  mallei  by  partic- 
ally  the  same  method  as  those  em- 
ployed in  the  preparation*  of  tuber- 
culin. The  organism  used  in  the  pre- 
paration of  Mallein  should  be  viru- 
lent. It  is  inoculated  into  flasks  of 
glycerine  bouillon  having  reaction  of 
3  and  incubated  at  a  temperature  of 
37°  C.  for  several  weeks.  The  cul- 
tures are  removed  from  the  incubator, 
heated  in  streaming  steam,  passed 
through  a  Burkefeld  filter,  the  filtrate 
concentrated,  preserved  and  put  up  in 
vials  ready  for  use. 

A  few  hours  after  the  injection  of 
mallein  into  a  horse  affected  with 
glanders,  a  severe  local  reaction  and 
a  rise  of  temperature  usually  follows. 
The  local  swelling  caused  by  the  mal- 
lein is  considered  by  some  to  be  as 
diagnostic  as  the  rising  temperature. 

Metcliniioif^s  Ph.ag'ocytic  Theory. 

The  term  "phagocyete"  is  given  to  any 
cell    capable   of   incorporating  bac- 
teria and  of  destroying  them  by  a 
process  of  digestion. 
Phagocytic  cells  comprise: — 

1.  Microphages,  polymorphonuclear 

leucocytes.  , 

2.  Macrophages   are   all   other  leuco- 

cytes, endothelial  cells  and  con- 
nective tissue  corpuscles  having 
phagocytic  power. 

When  animals  are  subjected  to  an 
irritant,  phagocytosis  occurs.  The 
leucocytes  are  attracted  by  chemo- 
taxis  to  the  zone  of  irritation  and 
envelop  the  irritating  substance. 

The  organisms  that  escape  from  one 
cell  are  seized  by  others,  but  if 
their  multiplication  is  excessive 
they  overpower  the  phagocytic 
leucocytes  and  invade  the  blood 
serum.  The  blood  serum  and  the 
body  fluids  are  likewise  bac- 
tericidal, due  to  the  disintegra- 
tion of  phagocytes — phag'olysis, — 
the  properties  of  these  cells  be- 
ing imparted  to  the  serum.  This 
property  is  due  to  two  constitu- 


178 


BACTERIOLOGY. 


ents  of  the  plasma.  The  one  (the 
specific  immune  body)  circulates 
In  the  plasma  and  resists  a  temp- 
erature of  100°  C.  The  other,  or 
"cytase"  (digestive  ferment),  de- 
rived from  the  disintegrated  pha- 
gocytes, corresponds  to  Buchner's 
"alexins"  and  Ehrlich's  "lysins." 
It  ie  destroyed  at  60°  C. 


AGGRESSINS. 

Certain  bacteria  may  be  injected  into 
an  animal  in  considerable  quantities 
without  producing  any  effect  other 
than  the  temporary  local  disturbance 
following  the  subcutaneous  admini- 
stration of  the  material. 

Certain  other  bacteria,  on  the  other 
hand,  as  the  bacillus  of  anthrax  or 
chicken  cholera,  may,  if  injected  even 
in  the  most  minute  dose,  give  rise  to 
a  rapid  fatal  septicemia.  Within  the 
same  species  fluctuations  in  virulence 
may  take  place,  depending  upon  a 
variety  of  influence,  but  variations  in 
the  susceptibility  of  the  inoculated 
subject  do  not  furnish  a  sufficient  ex- 
planation for  the  reaction  so  that  the 
explanation  must  be  put  down  to  the 
activities  of  the  bacteria  themselves. 

Pathogenic  bacteria  differ  from  non 
pathogenic  bacteria  in  their  power  to 
overcome  the  protective  mechanism  of 
the  animal  body,  and  to  proliferate 
within  it.  They  do  this  by  reason  of  a 
certain  definite  substance  which  they 
give  ofC  in  the  nature  of  the  secre- 
tion, which  protects  them  against 
phagocytosis.  These  substances  were 
named  by  Bail  "aggressins."  The  ag- 
gressins  are  probably  absent  in  test 
tune  cultures,  but  can  be  found  in  the 
animal  body  in  the  exudates  occur- 
ring about  the  sight  of  inoculation  in 
rapidly  fatal  infections.  Bail  was 
able  to  show  that  fatal  infections 
could  be  produced  in  animals  by  the 
Injection  of  sub  lethal  doses  of  bac- 
teria if  a  small  quantity  of  aggressin 
was  administered  at  the  same  time. 
He  believed  that  the  aggressin  para- 
lyzed the  phagocytic  and  other  pro- 
tective agencies  which  made  it  pos- 
sible for  the  bacteria  to  proliferate. 
He  further  showed  that  animals  were 


BACTERIOLOGY. 


179 


successfully  immunized  with  a§:gres- 
sins.  These  animals  were  not  only 
immune  themselves,  but  contained  a 
substance  in  their  serum  which  per- 
mitted the  passive  immunization  of 
other  untreated  animals. 

Bail's  theory  has  been  attacked  by  Was- 
serman.  Citren.  Wolfe  and  others. 
These  men  claim  that  much  of  the 
aggressive  character  of  Ball's  ex- 
udates is  due  to  their  containing  lib- 
erated bacterial  poisons  (endotoxin). 

Opsonins.  Wright  and  Douglas  demon- 
strated that  there  were  present  in 
blood  serum  and  the  body  fluids  cer^ 
tain  substances  that  had  the  power 
of  rendering  bacteria  susceptible  to 
phagocytosis.  These  substances  were 
termed  opsonia^s  (I  prepare  food  for). 
The  opsonins  act  chemically  upon  cer- 
tain substances  within  the  bacteria 
and  sensitize  them.  Phagocytosis  de- 
pends almost  wholly  on  these  specific 
opsonins  which  are  present  in  many 
normal  sera  for  the  various  bacteria. 
Its  presence  was  demonstrated  by 
washing  leucocytes  free  from  all  se- 
rum, when  they  refused,  except  in 
rare  cases,  to  take  up  bacteria.  Bac- 
teria which  have  been  placed  in  con- 
tact with  blood  serum  or  body  fluids 
and  thoroughly  washed,  will,  when 
placed  in  contact  with  leucocytes,  be 
taken  up  by  them.  Opsonins  may  be 
produced  in  animals  not  containing 
them  by  the  process  of  immunization. 
Opsonins  are  destroyed  at  about  60°  C. 
for  thirty  minutes.  They  will  remain 
active  for  several  days  at  0°  but  will 
deteriorate  rapidly  in  the  withdrawn 
blood  if  stored  at  a  temperature  of 
37°  C.  Many  opsonins  have  the  fea- 
tures of  agglutinins  and  precipitins, 
although  they  bear  some  points  of  re- 
semblance to  antitoxins  and  comple- 
ments. They  possess  a  haptophore 
group  with  which  they  combine  with 
the  bacteria,  and  a  functional  group, 
which  sensitizes  the  microorganisms 
for  phagocytosis. 

Opsonins  may  be  increased  in  the  serum 
of  normal  or  infected  individuals  by 
the  injection  of  heated  (60°)  cultures 
of  these  speciflc  etiological  micro- 
organisms. These  substances  are 
called   opsonogrens   or  vaccines  (seo 


180  BACTERIOLOGY. 


Bacterins)  and  are  extensively  used 
in  the  treatment  of  various  pus  in- 
fections,-due  to  the  staphylococci,  and 
also  in  tuberculosis  and  to  a  less  ex- 
tent 'in  pneumonia. 
Wright,  in  his  work,  makes  use  of  the 
so  called  opsonic  index  in  order  to 
estimate  the  changes  in  the  resist- 
ance of  a  patient  against  the  given 
infection. 

The  Determination  of  the  Opsonic  Index 

(in  order  that  the  concentration  of 
opsonins  in  an  individual  may  be 
recorded). 

1.  By  means  of  Wright's  capsule  col- 

lect blood  from  the  finger.  Seal 
the  capsule  at  both  ends;  allow 
the  blood  to  clot;  and  hasten  the 
separation  of  th^  serum  by  a  few 
revolutions  in  the  centrifuge. 

2.  Make  a  bacterial  emulsion  by  rub- 

bing up  a  few  loopfuls  of  a  24 
hour  slant  agar  culture  with  a 
little  physiological  salt  solution; 
this  emulsion  must  be  even. 

3.  Bleed  10  to  15  drops  from  the  ear 

or  finger,  directly  into  5  or  6  cc. 
of  a  normal  saline  solution  con- 
taining 1V2%  sodium  citrate.  Cen- 
trifugalize  for  5  or  6  minutes,  at 
the  end  of  which  time  the  cor- 
puscles at  the  bottom  of  the  tube 
will  be  covered  by  a  thin,  greyish 
pellicle  consisting  chiefly  of  leu- 
cocyte. Pipette  these  off  with  a 
capillary  pipette  (by  careful, 
superficial,  scratching  movements 
over  the  surface  of  the  buffy  coat 
forming  the  greyish  pellicle). 

The  serum,  the  bacterial  emulsion 
and  leucocytes  having  thus  been 
prepared,  the  test  is  carried  out 
as  follows: 

With  a  greased  pencil  make  a  mark 
upon  a  six  or  seven  inch  capillary 
pipette,  about  2  to  3  cm.  from  the 
end,  and  successively  draw  into 
the  pipette  up  to  the  mark,  cor- 
puscles, bacteria  and  serum, 
separating  them  from  one  another 
by  small  air  bubbles.  Equal  quan- 
tities of  each  having  thus  been 
secured,^  they  are  thoroughly 
mixed  by  repeatedly  drawing 
them  in  and  out  of  the  pipette 
upon  a  slide.    The  mixture  is  then 


BACTERIOLOGY. 


181 


drawn  into  the  pipette;  the  end 
is  pealed;  incubated  at  37i^°  for 
about  15  to  30  minutes. 

A  control,  or  normal  serum,  is  pre- 
pared and  treated  in  exactly  the 
same  way.  (The  normal  or  con- 
trol serum  is  obtained  by  a  •'Pool" 
or  mixture  of  the  sera  of  5  or  6 
supposedly  normal  individuals). 

After  incubation,  the  end  of  the 
pipette  is  broken  oft,  the  con- 
tents are  again  mixed,  and  smears 
are  made  upon  glass  slides  in  the 
ordinary  way,  and  stained  with 
Wright's  or  Jenner's  stain,  and 
the  number  of  bacteria  contained 
in  each  leucocyte  is  counted.  The 
contents  of  about  80  to  100  cells 
are  usually  counted  and  averages 
taken.  This  average  number  of 
bacteria  in  such  leucocytes  is 
spoken  of  as  the  phagocytic  in- 
dex. The  phagocytic  index  of  the 
iested  serum  divided  by  that  of 
the  normal  "pool"  serum,  gives 
the  opsonic  index.  (Suppose  the 
leucocytes  of  the  infected  indi- 
vidual take  up  an  average  of  five 
bacteria.  In  this  case,  the  pha- 
gocytic index  is  said  to  be  five. 
Again,  suppose  the  leucocytes  of 
the  normal  individual  take  up  15 
bacteria.  The  phagocytic  indejc 
in  this  case  would  be  15.  The 
opsonic  index  of  the  infected  in- 
dividual would  therefore  be  0.33 
+,  as  the  normal  individual  pha- 
gocytic index  is  taken  as  the  de- 
nominator of  a  fraction  and  the 
phagocytic  index  of  the  infected 
individual  as  enumerator,  there- 
fore, it  would  be  5/15  or  1/3). 

The  opsonic  index  would  therefore 
seem  a  fair  indication  as  to  the 
resistance  of  the  particular  indi- 
vidual to  the  infecting  micro- 
organism. By  the  judicious  use 
of  vaccines,  the  opsonic  index 
may  be  raised  to  at  least  1.0  or 
even  more,  showing  that  the  leu- 
cocytes are  actively  phagocytic 
and  opsonins  increased  in  concen- 
tration of  the  blood  serum.  In 
such  cases,  recovery  will  be  indi- 
cated. 


182  BACTERIOLOGY. 


The  opsonic  index  gives  a  fair  idea 
as  to  the  resistance  of  an  indi- 
vidual to  an  infecting  micro- 
organism. 

Virulent  bacteria  are  not  phago- 
cytized.  Virulent  streptococci 
and  pneumococci  are  not  as  easily 
taken  up  as  the  non  virulent 
forms.  It  would  seem  from  this 
that  some  toxic  or  poisonous  sub- 
stance produced  by  the  bacteria  is 
antagonistic  to  opsonins,  or  it 
may  be  that  an  anti  opsonin  is 
formed. 

The  presence  of  opsonins  in  the  body 
fluids  of  an  animal  is  not  absolute 
proof  that  such  animal  is  highly 
resistant  to  infection.  The  re- 
sistance depends  upon  the  activ- 
ity of  the  phagocytes,  and  in 
certain  cases  where  the  opsonins 
are  high  in  concentration  the  pha- 
gocytes are  not  active.  In  certain 
cases  the  reverse  is  true,  and  here 
the  opsonins  and  phagocytosins 
are  of  the  utmost  importance  dur- 
ing the  immunity  of  the  in- 
dividual. 


LEUCOCYTIC  EXTRACT. 

Hiss  conceived  the  plan  of  injecting 
into  infected  subjects  the  substances 
jthat  compose  the  chief  cells,  or  all 
the  cells  usually  found  in  extradites, 
in  the  most  diffusible  forms  and  as 
little  changed  by  manipulation  as  pos- 
sible, in  order  that  the  living  leuco- 
cytes which  exert  the  protective  ac- 
tion against  bacterial  infection  might 
be  considerably  reinforced  as  directly 
as  possible  with  a  further  supply  of 
the  weapons  that  they  use  against 
the  microorganisms. 

Hiss  also  assumed  that  extracts  from 
the  leucocytes  would  be  more  effica- 
cious than  the  living  leucocytes  them- 
selves, in  that  if  they  were  diffusible 
they  would  be  distributed  impartially 
to  all  parts  of  the  body  by  the  cir- 
culatory system.  In  this  way,  quick 
absorption  would  relieve  the  tired  out 
leucocytes  and  would  also  protect  by 
any  toxin-neutralizing  or  other  power 
they  might  possess,  the  cells  of  high- 
Iv  specialized  functions. 


BACTERIOLOGY. 


183 


Method  of  obtaining'  tliese  substances 

(for  animal  experiments  and  treat- 
ment of  human  subjects).  Rabbits  of 
1500  gms.  weight  or  over  are  injected 
intraplurally  with  aleuronat  (prepared 
by  making  a  3%  solution  of  starch 
and  meat  extract  broth,  without  heat- 
ing; after  the  starch  has  gone  into  a 
thorough  emulsion,  5%  of  powdered 
aleuronat  is  added;  after  thorough 
mixing,  boil  for  5  minutes;  fill  into 
sterile  potato  tubes  in  quantities  of 
20  cc.  in  each  tube;  sterilize  in  the 
autoclave). 
10  cc.  of  the  mixture  is  injected  into 
each  plural  cavity,  in  the  intercostal 
spaces  at  the  level  of  the  end  of  the 
sterum,  in  the  anterior  axillary  line, 
taking  care  that  the  lungs  are  not 
punctured.  At  the  end  of  24  hours  a 
copious  cellular  exudate  will  have  ac- 
cumulated in  the  plural  cavities.  Kill 
the  animal  with  chloroform  and  under 
rigid  sterility  open  the  anterior  chest 
wall  and  pipette  the  exudate  into 
sterile  centrifuge  tubes.  Centrifugal- 
ize  immediately  before  clotting  can 
take  place;  decant  the  supernatent 
fluid.  Add  to  the  leucocytic  sediment 
about  2  cc.  of  sterile,  distilled  water, 
and  make  into  an  emulsion  by  means 
of  a  platinum  spatula.  Make  smears, 
stain  with  Jenner's  blood  stain  and 
examine  for  possible  bacterial  con- 
taminations. It  is  well  to  test  for 
contamination  by  the  culture  method. 
Now  add  to  each  tube  about  20  vol- 
umes of  sterile,  distilled  water  to  one 
volume  of  the  sediment;  set  aside  in 
the  incubator  for  8  hours.  Test  again 
for  sterility.  Store  in  the  refriger- 
ator, where  further  extraction  takes 
place,  until  the  extract  is  used.  Hiss 
and  Zinsser  have  injected  the  extract 
subcutaneously  as  treatment  in  cases 
of  epidemic  cerebro-spinal  meningitis, 
in  lobar  pneumonia,  in  staphylococci 
infections  and  in  erysipelas  with 
beneficial  results. 

ANAPHYLAXIS  OR  HYPER 

SUSCEPTIBILITY. 

When  a  foreign  proteid  is  introduced 
into  the  body,  after'  a  time  there  will 
appear  a  specific  hypersusceptibility 


184  BACTERIOLOGY. 


of  the  animal  for  this  proteid.  If, 
after  a  definite  interval,  a  second  in- 
jection of  the  same  substance  is  given, 
violent  symptoms  of  illness  will  be 
produced,  and  often  death. 

As  early  as  1893,  Behring  noticed  that 
animals  highly  immunized  against 
diphtheric  toxin  would  occasionally 
show  marked  susceptibility  to  in- 
jections of  small  doses  of  the  toxin. 

Wolfe  and  Esiner  believe  that  all  cells 
and  proteid  material  contain  a  toxic 
substance  which  is  characterized  by 
its  inability  to  produce  a  neutralizing 
antibody  when  injected  into  animals. 
The  first  injection  produces  a  lysin 
for  the  proteid  injected,  which  pos- 
sesses the  power  of  liberating  such 
poisons  from  the  complex  molecules; 
consequently,  when  a  second  injection 
is  given  there  is  a  rapid  liberation  of 
the  toxic  fraction,  and  an  injury  to  the 
animal  results.  This  view  has  been 
supported  experimentally  by  Vaughn 
and  Wheeler,  who  have  been  able  to 
extract  from  various  proteids  toxic 
substances  which  give  rise  in  animals 
to  symptoms  not  unlike  those  of  typ- 
ical anaphylaxis. 

PATHOGENIC  MICRO-ORGANISIMS, 
THE  STAPHYLOCOCCI 
(MICROCOCCI). 

The  Staphylococcus  Pyogrenes,  Anretis 
and  Alhus.  These  organisms  are  found 
to  cause  infections,  such  as  boils,  ab- 
scesses, osteomyelitis,  pyemia,  etc., 
throughout  the  world.  These  organ- 
isms stain  readily  in  pus  with  aniline 
dyes,  and  the  simple  sowing  upon  or-- 
dinary  media  is  usually  suflflcient  for 
cultures,  but,  if  pure  cultures  are 
wished,  plating  should  be  resorted  to. 

Man  seems  to  be  considerably  more  sus- 
ceptible to  staphylococci  infections 
than  other  animals.  The  virulence  of 
the  organisms  varies  and  is  increased 
by  successive  passage  through  an- 
imals of  the  same  species,  but  re- 
mains unaltered  for  animals  of  other 
species.  Virulent  cultures,  injected 
into  the  peritoneal  cavity  of  animals, 
may  kill  in  from  48  hours  to  a  week, 
or    longer,    with    abscess  formation, 


BACTERIOLOGY. 


185 


especially  in  the  kidney.  Malignant 
or  ulcerative  endocarditis  has  been 
experimentally  produced;  likewise, 
osteomyelitis.  Simply  rubbing-  the 
virulent  cultures  in  the  skin  of  man 
often  produces  furuncles. 
Immunization  can  be  secured  by  in- 
jections of  the  dead  or  live  cocci  in 
graduated  doses.  The  serum  posses- 
ses slight  bactericidal  and  agglutin- 
ated properties,  also  a  high  degree  of 
opsonic  power.  The  serum  is  protec- 
tive only  when  used  slightly  before 
or  along  with  the  injection  of  the 
organism,  hence, of  little  value.  Active 
immunization  is  extensively  practiced 
with  autogenous  strains  of  the  organ- 
isms. 

Tlie  variety  aureus  is  spherical  in  shape. 
On  solid  media  it  is  found  singly,  in 
pairs  or  in  rows  of  three  or  four,  but 
generally  in  irregular  groups,  like 
bunches  of  grapes.  In  liquid  media, 
the  single  and  paired  forms  are  most 
frequent.  It  is  .gram  positive.  Temp- 
erature range  of  growth  is  10°  to  43°. 
Optimum  about  30°.  Grows  readily  on 
all  culture  media  of  a  slightly  alka- 
line reaction.  On  agar,  after  24  hours, 
small,  round,  greyish  white  or  yellow 
colonies  appear.  The  characteristic 
orange  yellow  pigment  may  not  ap- 
pear until  later.  In  broth,  growth  is 
rapid  with  diffused  clouding,  with  a 
thin  pellicle  and  a  heavy  sediment 
after  several  days.  In  gelatine,  col- 
onies sink  into  cups  of  liquefaction. 
Liquefaction  is  due  to  a  thermol  label 
ferment  substance  called.  Gelatinis. 
Milk  is  coagulated  in  three  or  four 
days  time.  Potato,  abundant  growth 
not  as  moist  or  smooth  as  on  agar. 
Acid  but  no  gas  is  produced  in  dex- 
trose, lactose  and  saccharose  media. 
Presence  of  fatty  acids  produces  char- 
acteristic odor  of  cultures.  Pigment 
appears  in  the  aerobic  but  not  in  the 
anaerobic  culture.  The  pigment  is  in- 
soluble in  water  but  soluble  in  alcohol, 
chloroform,  ether  and  benzol.  The 
toxins  are  largely  intracellular.  In 
,  the  more  virulent  strains  grown  in 
moderately  alkaline  broth,  a  thermol 
label  hemolytic  substance  can  be  ob- 
tianed  by  filtration  through  porcelain 
filters.      Another    toxic    substance  is 


186  BACTERIOLOGY. 


found  that  causes  the  death  of  leu- 
cocytes (leucocidia).  This  toxin  is 
less  stable  than  the  one  mentioned 
above  (staphylo-haemolysin). 

Staphylococci  are  more  resistant  than 
are  the  other  non  spore-bearing  bac- 
teria. An  hour  or  more  at  60*  is 
necessary  to  kill  watery  suspensions; 
70°  is  necessary  to  kill  in  10  minutes. 
Resistance  is  much  greater  if  organic 
material  is  present.  Low  tempera- 
tures have  little  effect;  thirty  per 
cent  have  survived  30  minute  ex- 
posure to  liquid  air.  They  resist  dry- 
ing and  direct  sunlight  to  a  marked 
degree.  They  may  be  found  in  pulp 
that  has  been  dried  for  several 
months.  To  the  germicides,  especial- 
ly in  the  presence  of  organic  matter, 
they  are  more  resistant  than  other 
vegetative  bacteria. 

The  variety  albus.  This  organism  dif- 
fers from  the  pyogenes  aureus  simply 
In  the  absence  of  the  golden  color. 
Morphologically,  culturally  and  path- 
ogenically,  it  is  identical.  Its  toxin 
and  enzyme  producing  power  is  gen- 
erally less  than  the  aureus,  otherwise, 
its  biological  relationship  is  so  close 
RS'  to  be  demonstrated  by  its  agglu- 
tinations in  the  aureus  immune. 

The  Staphylococcns,  Epidermidis  Alhus 
was  described  by  Welch  and  may  give 
rise  to  stitch  abscesses.  It  is  merely 
one  of  the  non  pathogenic  forms  of 
the  staphylococcus  pyogenes  albus. 

The  Staphylococcus  Fyogfenefl  Citrens 
differs  from  the  staphylococcus  pyo- 
genes aureus  in  its  bright  yellow  or 
lemon  colored  pigment.  It  may  be  as 
pyogenic,  biit  is  less  often  found  in 
connection  with  pathological  lesion. 

A  large  number  of  staphylococci  differ- 
ing from  those  mentioned  above  have 
been  observed.  Few  of  these  have  any 
pathological  significance  and  so  far  as 
known  they  have  no  toxin  producing 
properties.  They  are  frequently  met 
with  as  contaminations  in  the  course 
of  bacteriological  work. 

Micrococcus  Tetragfemis  was  discovered 
in  1881  by  Gaffney  in  the  pus  of 
tubercular  patients.  Stained  smears 
of  the  pus  containing  the  micrococcus 
show  them  in  the  form  of  tetrads 
larger  than  the  staphylococci.  They 


BACTERIOLOGY. 


187 


are  flattened  along  their  adjacent  sur- 
faces and  are  surrounded  by  a  thick, 
halo  like  capsule.  It  stains  easily 
with  the  usual  aniline  dyes,  also  by 
Gram's.  It  grows  on  all  the  ordinary 
media.  On  agar,  the  colonies  first  ap- 
pear as  transparent  spots  which  later 
become  greyish  white,  but  are  al- 
ways more  transparent  that  the  other 
staphylococci  cultures. 
On  gelatine,  growth  is  slow;  no  lique- 
faction. Broth,  is  clouded.  Potato, 
growth  is  whrte  and  moist  showing 
a  tendency  to  confluence.  Milk,  co- 
agulated. Litmus  milk,  acid  forma- 
tion. 

In  man,  the  organism  is  probably  with- 
out any  pathogenic  significance  when 
found  in  the  sputum  or  saliva.  In  a 
few  isolated  cases,  it  may,  however, 
be  the  cause  of  abscess  formation. 
Bezancon  isolated  the  organism  from 
a  case  of  meningitis  and  Forneaca  re- 
ported a  case  of  tetragenus  septi- 
cemia. 

The  organism  Is  seldom  found  in  con- 
nection with  disease,  but  it  is  often 
found  in  considerable  numbers  in 
sputum  examined  for  pneumococci  or 
tubercle  bacilli. 

Experimentally,  the  organism  is  especi- 
ally (pathogenic  for  Japanese  mice.  If 
injected  subcutaneously,  death  occurs 
in  three  or  four  days.  Grey  mice, 
rats,  guinea  pigs  and  rabbits  are  less 
susceptible  and  show  only  a  localized 
reaction  at  the  point  of  inoculation. 


THE  STREPTOCOCCI. 

The  Streptococci  Pyogrenes  grow  in  long 
chains  and.  ferment  lactose,  saccha- 
rose and  salicin  but  do  not  coagulate 
milk.  This  group  comprises  most  of 
the  streptococci  which  cause  suppur- 
ative lesions  or  severe  systemic  in- 
fections. 

Streptococcic  infections  are  endemic 
among  all  races  and  under  all  social 
conditions.  They  are  more  frequently 
found  in  the  human  being,  however, 
than  in  horses,  cattle  and  laboratory 
animals.  The  period  of  incubation  is 
probably  about  one  to  three  days. 

Streptococci  seem  to  be  always  present 
on  the  exposed  surface  of  the  body 


188  BACTERIOLOGY. 


and  are  capable  of  causing  infection 
should  any  local  lowered  resistance 
occur.  The  symptoms  of  septicemia 
are  a  rapid  rise  in  temperature  to  a 
105"  P.  or  over,  chills,  rapid,  irregular 
and  weak  pulse,  respiration  labored, 
may  be  vomiting,  and  constipation  or 
diarrhea.  Headache  more  or  less  se- 
vere, sometimes  delirium.  Death  may 
occur  in  two  or  three  days  or  within 
a  week.    Mild  cases  may  recover. 

Death  from  septicemia  causes  the  body 
to  putrefy  rapidly.  The  glandular 
organs,  especially  the  spleen,  tend  to 
be  swollen  and  soft,  and  parenchy- 
matous degenerations  are  found  to  a 
greater  or  less  extent.  The  endo- 
thelium of  the  heart  and  vessels  is 
blood  stained,  which  is  a  character- 
istic feature  of  streptococcic  septi- 
cemia. Bronchitis  and  broncho  pheu- 
monia  are  usually  found. 

Erysipelas  is  an  inflammation  of  the 
skin  and  sometimes  of  the  mucous 
membrane  brought  about  by  the  strep- 
tococci. The  area  involved  is  definite- 
ly outlined.  Oedema  ^nay  be  white 
marked  where  the  skin  covers  loose 
pigment.  Fever  with  its  accompani- 
ment is  present.  There  may  be  vomit- 
ing, constipation  or  diarrhea,  severe 
headaches  or  delirium.  Death  may 
occur  without  any  apparent  compli- 
cation or  death  may  follow  meningitis, 
pericarditis  or  nephritis. 

Superficial  cutaneous  infections  are  met 
with  and,  if  mild,  may  be  similar  to 
the    localized    abscesses    caused  by 

'  staphylococci.  But  if  severe  the  in- 
fection is  followed  by  rapid  spreading 
oedemia,  lymphangitis,  severe  sys- 
temic manifestations,  a  grave  cellu- 
litis, often  threatening  life  and  re- 
quiring energetic  surgical  interference. 

The  respiratory  organs  may  be  invaded 
leading  to  bronchitis,  pneumonia  and 
empyema.  It  may  be  present  as  a 
secondary  infection  in  tuberculosis. 
The  infection  of  the  lung  and  plura 
frequently  leads  to  pericardial  in- 
volvement. 

Streptococci  may  invade  bones  and  pro- 
duce a  severe  form  of  osteomyelitis. 
If  occurring  in  the  mastoid  bone,  it 
may  lead  to  meningitis. 


BACTERIOLOGY. 


189 


In  the  throat  and  mouth,  pharyngitis 
may  be  produced  together  with  the 
tonsillitis  that  may  be  easily  mistaken 
for  the  diphtheria.  The  inflamma.tory 
throat  present  in  scarlatina  is  almost 
always  due  to  the  streptococcus.  A 
secondary  infection  of  this  organism 
following  diphtheria  is  a  frequent  and 
serious  complication, 

Str»eptococcic  throat  infections  have  re- 
cently appeared  as  epidemics.  Sev- 
eral small  epidemics  took  place  in 
England.  Severe  epidemics  have  ap- 
peared in  this  country;  one  in  Boston, 
one  in  Chicago  and  another  in  Balti- 
more. Investigation  traced  the  infec- 
tion in  the  majority  of  these  cases 
to  a  single  milk  supply.  Those  sec- 
ondary cases  occurred  by  contact. 
Complications,  such  as  suppurative 
adenitis,  otitis,  erysipelas,  peritonitis 
and  septicemia,  were  frequent.  A' 
capsulated  hemolytic  streptococcus 
was  found  in  each  epidemic. 

Prom  any  local  process,  streptococci 
may  pass  into  the  circulation,  causing 
sepsis.  The  septicemia  occurring  dur- 
ing the  puerperium  is  often  caused  by 
this  organism.  Streptococci  have  been 
found  in  appendiceal  abscesses. 

Secondary  foci  in  the  viscera  may  take 
place  and  lead  to  pyemia,  if  localized 
upon  the  valves  of  the  heart,  septic 
endocarditis  results.  All  forms  of 
streptococcic  infection,  whether  acute 
or  chronic,  is  followed  by  a  high 
mortality.  The  diagnosis  in  these 
cases  is  usually  made  by  means  of 
blood  cultures  in  plain  broth  or  other 
suitable  media. 

The  streptococci  vary  somewhat  in  size. 
In  shape  they  may  be  rounded  or  oval, 
or  with  one  aspect  flattened  when  they 
occur  in  pairs.  The  chains  formed 
may  be  long  or  short,  and  a  grouping 
into  pairs  is  quite  frequent,  even 
when  the  organism  is  formed  into 
chains.  The  organism  is  non-motile 
and  without  spores.  It  stains  with 
the  ordinary  aniline  dyes,  gram  posi- 
tive, temperature  range  from  15  to  45, 
the  optimum  about  37°.  The  organ- 
ism is  aerobic  and  facultative  anae- 
robic. A  strict  anaerobic  species  has 
been  said  to  have  been  isolated  from 
feces.      Culture     media     should  be 


190  BACTERIOLOGY. 


slightly  alkaline  in  reaction.  Acid 
is  produce(J  which  has  a  inhibitory 
action  upon  its  growth.  Acids  are 
formed  from  the  monosaccharides 
lactose,  saccharose  and  salicin.  Gas 
production  is  negative.  Nitrates  are 
reduced  to  nitrites  in  some  cases. 
Hydrogen  sulphide  is  produced  by  a 
group  called  streptococcus  faecalis. 
No  pigment  except  a  slight  brownish 
tinge  in  some  gelatin  cultures.  It  is 
actively  hemolytic  which,  however,  is 
lost  on  cultivation.  The  toxic  pro- 
ducts of  the  organisms  have  been 
deeply  investigated  without  any  defin- 
ite facts  discovered.  On  agar,  a  visi- 
ble growth  appears  in  18  to  24  hours, 
as  small,  round,  translucent  colonies, 
with  even  or  notched  borders,  center 
thick  and  margins  thin.  The  colonies 
show  a  tendency  to  remain  discreet. 
In  nutrient  broth,  the  long  chain 
varieties  produce  granular  deposits 
or  small  flocculi  or  large  flakes  at  the 
bottom  and  along  the  sides  of  the 
tube,  leaving  the  remainder  of  the 
broth  clear.  Certain  few  long  chain  va- 
rieties produce  a  uniform  cloudiness. 
The  short  chain  varieties  generally 
produce  a  cloudiness  in  the  medium 
which  remains  so  for  a  number  of 
days  even  though  a  fine,  granular  de- 
posit accumulates  at  the  bottom  of 
the  tube.  The  gelatin  colony  is  the 
same  as  that  of  the  agar.  Stab  cul- 
tures are  at  first  finely  granular  fili- 
form which  later  become  beaded  and 
may  assume  a  brownish  color.  The 
gelatin  is  not  liquefied.  Milk  becomes 
strongly  acid  and  coagulation  may 
take  place.  On  potato,  no  growth  re- 
sults except  in  some  cases  when  there 
seems  to  be  an  invisible  growth. 
LoeflEier's  blood  serum  is  a  favorable 
medium. 

The  streptococci  will  die  out  rapidly  in 
cultures  due  to  the  accumulation  of 
their  own  products.  The  organism 
may  be  found  alive  after  several 
weeks  or  months  at  room  temperature 
in  pus,  blood  or  sputum.  The  thermal 
death  point  is  45°.  Direct  sunlight 
Will  kill  within  a  few  hours. 

Immunity  following  the  recovery  from 
natural  streptococcic  infections  is 
very  slight,  if  any,  and  never  of  a 


BACTERIOLOGY. 


191 


permanent  nature.  Septicemias  once 
established  are  generally  fatal  and 
erysipelas  can  recur  frequently.  Ac- 
tive immunity  may  be  produced  in 
rabbits,  goats,  horses  and  other 
domestic  animals  by  treatment  with 
gradually  increased  doses  of  the  cul- 
tures. The  bacterial  substances,  op- 
sonins, agglutinins  and  precipitins 
have  been  demonstrated  in  the  im- 
mune serum,  which,  however,  shows 
no  therapeutic  success. 
The  Streptococcus  Mitis  is  a  saprophytic 
type  of  the  mouth,  showing  the  same 
culture  characteristics  as  the  strep- 
tococcic pyogenes,  but  grows  in  short 
chains. 

The  Streptococcus  Anglnosns  is  a  type 
found  frequently  in  scarlet  fever 
throats  and  differs  only  from  the 
streptococcus  pyogenes  in  coagulating 
milk. 

The  Streptococcus  Salivarius  is  a  short 
chain  type  frequently  found  in  the 
mouth,  rarely  pathogenic,  which  fer- 
ments lactose,  saccharose  and  rafR- 
nose.    It  coagulates  milk. 

The  Streptococcus  Fecalis  is  a  short 
chain  type  found  normally  in  the  in- 
testine and  is  occasionally  pathogenic, 
which  ferments  lactose,  saccharose 
and  mannlte. 

The  Streptococcus  Eg.uinus  is  a  short 
chain  type  found  normally  in  horse 
dung  and  is  never  pathogenic.  It 
ferments  lactose. 

The  Streptococcus  Mucosus.  This  or- 
ganism was  described  by  Howard  and 
Perkins  in  1901.  It  was  isolated  by 
Schottmuller  from  cases  of  para- 
metritis, peritonitis,  meningitis  and 
phlebetis.  Some  have  claimed  it  to 
be  the  cause  of  a  variety  of  lesions; 
others  describe  it  as  a  harmless  or- 
ganism of  the  normal  mouth.  Morph- 
ologically, it  shows  a  tendency  to  pro- 
duce chains.  On  solid  media,  it  often 
appears  as  a  diplococcus.  It  is  cap- 
sulated  and  is  therefore  similar  to 
the  pneumococcus  but  does  not  have 
the  typical  lancet  shape.  The  fact 
that  it  ferments  inulin  media  and  on 
account  of  its  agglutinating  proper- 
ties, it  might  more  accurately  be 
placed,  in  the  group  of  pneumococci 
than  in  the  group  of  streptococci. 


192  BACTERIOLOGY. 


The  Poyntou  and  Paine  Streptococcus 

(Rheumaticus).  A  diplococcus  iso- 
lated from  eight  cases  of  acute  rheu- 
matic fever  and  with  which  Poynton 
and  Paine  produce  lesions  in  rabbits 
which  they  considered  typical  of  rheu- 
matism. The  organism  was  recovered 
from  the  blood  from  the  pericardial 
fluid  or  the  tonsil  of  the  patients.  It 
was  described  as  a  minute,  gram 
negative  diplococcus  growing  in  acid 
media  under  anaerobic  conditions,  but 
would  grow  under  aerobic  conditions. 
Attemps  to  confirm  their  work  have 
met  with  negative  results.  Rosenow 
»  has,  however,  reported  a  streptococcus 
isolated  from  the  joints  of  articular 
rheumatic  patients  and  has  been  able 
to  produce  non-suppurative  arthritis, 
endocarditis  and  pericarditis  in  rab- 
bits. He  describes  the  organism  as 
intermediate  in  character  between  the 
streptococcus  viridans  and  the  strep- 
tococcus hemolytica. 

DIFI^OCOCCnS  PNEUMONIA. 

(Pneumococcus,  Diplococcus  lanceolatus. 
Micrococcus  Pneumonia,  Streptococ- 
.  cus  Pneumonia). 

The  occurence  of  a  diplococcus  in  a 
large  majority  of  cases,  especially  of 
the  lobar  type  of  pneumonia,  has 
caused  this  coccus  to  be  regarded  as 
practically  specific.  About  90%  of  all 
cases  of  acute,  lobar  pneumonia  is 
caused  by  the  pneumococcus,  the  re- 
mainder being  due  to  streptococci  in- 
fluenza, bacillus.  Friedlander's  bacilli 
and  exceptionally  to  other  micro- 
organism's. Lobular  pneumonia  is  also 
caused  by  the  pneumococcus  with  al- 
most equal  regularity.  The  incuba- 
tion period  of  this  organism  is  two 
or  three  days.  The  onset  of  the  dis- 
ease is  marked  by  a  chill,  pain  and  a 
rise  in  temperature.  The  respirations 
become  frequent.  The  fever  runs  be- 
tween 102°  and  105°  F.  for  five  to  ten 
days  and  then  in  favorable  cases 
terminates  by  a  sudden  drop  to  normal 
within  a  few  hours. 

The  pathological  findings  are  (first 
stage)  congestion  and  oedema  of  the 
lungs,  followed  by  (second  stage)  the 
lung  becoming  solid,  airless  and  ot 
a  dark  red  color,  the  alveoli  showing 


BACTERIOLOGY.  193 


microscopically,  a  fibrous  exudate 
with  large  numbers  of  red  cells,  some 
leucocytes  and  desquamated  epithe- 
lium; (third  stage)  the  lung  becomes 
slightly  softer  and  of  a  grey  color, 
microscopically  the  red  cells  degen- 
erate and  leucocytes  are  increased  in 
number.  (fourth  stage)  Resolution 
takes  place  by  liquefaction  and  ab- 
sorption of  the  contents  of  the  alveoli) 
and  the  entrance  of  air. 
I  Death  occurs  from  toxemia  or  compli- 
cations, such  as  endocarditis,  menin- 
gitis, etc. 

For  animals,  the  pathogenic  properties 
of  the  pneumococcus  varies.  Natural 
infection  is  not  common.  Mice  and 
rabbits  are  most  susceptible  to  arti- 
ficial infection;  while  guinea  pigs, 
dogs,  rats  and  cats  are  more  resistant. 
Birds  are  nearly  immune  by  reason 
of  their  high  temperatures.  Subcu- 
taneous or  intraperitoneal  injections 
of  the  virulent  organism  from  cul- 
tures or  sputum  kill  mice  and  rabbits 
by  the  development  of  septicemia  and 
peritonitis.  The  virulence  of  the 
pneumococcus  may  be  increased  by 
passage  through  susceptible  animals 
until  an  extremely  small  dose  would 
kill  a  mouse.  The  virulence  of  the 
cultures  obtained  from  man  may  vary 
considerably  in  their  virulence  for 
animals. 

The  organism  appears  to  be  a  common 
inhabitant  of  the  respiratory  tract, 
acquiring  virulence  only  under  some 
special  condition  that  lowers  the  gen- 
eral vitality,  and  gaining  entrance 
through  the  respiratory  mucosa,  and 
during  the  disease,  it  is  frequent  to 
find  positive  blood  cultures;  a  fact 
which  accounts  for  the  development 
of  complications,  as  meningitis  and 
endocarditis.  The  toxemia  results 
probably  from  lysis  of  the  organism 
and  it  has  been  shown  that  autolysis 
of  cultures  in  salt  solution  gives  rise 
to  a  soluble  toxic  portion  and  an  in- 
soluble toxic  portion. 

Immunity  can  be  shown  to  exist  after 
an  attack  for  a  short  time  only. 

Specific  therapeutic  agents,  such  as  anti 
pneumococci  sera,  vaccines  of  dead 
cultures  and  autolysates  and  leuco- 
cyte extracts  have  been  tried  with 
some   promise   of   results.     No  one 


194  BACTERIOLOGY. 


method,  however,  has  been  applied 
sufficiently  with  success  enough  to 
warrant  general  adoption.  In  the 
Ksputum,  a  Gram  stained  specimen  is 
sufficient  to  detect  the  diplococcus, 
but  positive  identification  mi^st  be 
made  by  culture.  Culture  medium 
made  rich  by  the  addition  of  blood 
serum  from  man  or  animals  is  used. 

Inoculations  are  made  from  the  blood 
organs  or  sputum.  Sputum  injected 
into  white  mice  or  rabbits  will  often 
cause  a  fatal  septicemia,  and  the  or- 
ganisms may  then  be  obtained  in  pure 
culture  from  the  heart's  blood.  The  or- 
ganisms appear  in  pairs,  as  oval  or 
lancet  shape  cocci,  with  their  con- 
tiguous surface  flattened  and  the 
distal  ends  pointed.  The  organism 
may  vary  from  this  type  to  spherical 
or  short  bacillary  form.  The  organ- 
ism may  also  appear  singly  or  in 
chains  of  a  length  usually  not  more 
than  about  six  or  eight  individuals. 
Well  developed  capsules  envelope  the 
single  organism,  the  pairs  or  the 
chains.  There  are  no  spores  or 
flagella.     The   organism   stains  with 

»  the  ordinary  aniline  dyes  and  is  Gram 
positive.  The  temperature  range  is 
from  25**  to  37*'.  It  is  both  aerobic 
and  anaerobic  and  grows  in  a  slightly 
alkaline  media.  Glycerine,  nutrose 
and  dextrose  media  are  favorable  to 
their  growth.  On  agar,  small,  trans- 
parent, finally  granular  colonies  ap- 
pear. On  a  serum  or  ascitic  fluid 
agar,  the  colonies  are  slightly  larger 
and  more  opaque.  Broth  is  slightly 
but  uniformly  clouded.  Milk  is  acidi- 
fied and  coagulated.  On  potato,  a 
growth  occurs  but  is  invisible.  Fer- 
mentation with  acid  production  takes 
place  in  the  majority  of  carbohydrates, 
even  inulin.  On  blood  agar,  a  green- 
ish zone  appears  about  the  growth, 
but  no  clear  zone  of  hemolysis  as  ap- 
pears in  the  growth  of  streptococci. 
The  differentiation  from  other  strepto- 
cocci is  sometimes  difficult  but  the  fol- 
lowing characters  are  important  dis- 
tinguishing features: — the  lanceolate 
shape;  the  capsule;  fermentation  of 
inulin;  absence  of  hemolytic  power; 
agglutination  in  anti  pneumococcic 
serum;  susceptibility  to  lysis  by  the 


BACTERIOLOGY. 


195 


action  of  bile  salts.  Acid  is  a  char- 
acteristic product  and  if  allowed  to 
accummulate,  rapidly  kills  the  organ- 
ism. The  toxic  products  are  closely 
united  with  the  cell  bodies  and  only 
released  when  the  cells  are  broken  up. 
The  thermal  death  point  is  52°.  Light 
is  a  very  efficient  germicide  unless 
protected  in  thick  masses  of  sputum. 
Desiccation  is  resisted  well  in  the 
sputum  or  in  blood  of  infected  an- 
imals. The  ordinary  germicides,  if 
used  in  their  usual  strength,  will  kill 
the  organism  in  a  few  minutes. 

THE  MICROCOCCUS  INTBACEZiIkU- 
IkABIS  MENINGITIBIS. 

In  1887,  Weichselbaum  discovered  a 
micrococcus  in  the  exudate  of  cerebro- 
spinal meningitis  and  called  it  Diplo- 
coccus  Intracellularis  Meningitidis 
after  obtaining  it  in  pure  culture  and 
studying  its  characteristics.  He  suc- 
ceeded also  in  obtaining  the  diplo- 
coccus  from  the  nasal  secretion  of 
the  individual  sick  from  the  disease. 
Albreth  and  Ghon  (1901)  demon- 
strated the  organisms  in  healthy  in- 
dividuals. It  is  now  believed  that  the 
organism  is  not  infrequently  present 
in  the  nasal  cavities.  The  respiratory 
tract  through  winter  and  spring  pre- 
sents a  place  of  infection  and  where 
an  increase  in  the  virulence  of  the 
organisms  take  place.  Meningitis, 
in  some  cases,  will  follow  an  infection 
of  the  nasal  mucous  membrane  but 
not  in  others.  Why  this  is  so  is  not 
yet  known.  Infected  persons,  together 
with  the  material  recently  soiled  by 
the  nasal  secretions,  are  dangerous. 

The  organism  does  not  show  marked 
pathogenicity  for  adult  animals.  It  is 
most  pathogenic  for  mice  and  guinea 
pigs,  less  so  for  rabbits  and  dogs. 
Large,  subcutaneous  injections  in 
animals  cause  death.  Mice  injected 
into  the  plural  or  peritoneal  cavity 
usually  become  sick  and  die  within 
36  to  48  hours.  In  man,  the  most 
marked  lesions  occur  at  the  base  of 
the  brain.  The  cord  is  also  infected. 
The  exudate  formed  varies  from  a 
slightly  turbid,  serous  fluid  to  a  thick, 
fibrinous  consistency.  In  chronic 
cases,  encephalitis  and  dilatation  of 
the  ventricles  may   take  place.  Oc- 


196  BACTERIOLOGY. 


casionally,  secondary  inflammation  of 
the  nasal  cavities  and  their  accessory 
sinuses,  catarrhal  inflammations  of 
the  middle  ear,  acute  bronchitis  and 
pneumonia,  may  take  place.  Eisner 
examined  the  blood  during  the  early 
days  of  the  disease  in  forty  cases  and 
found  the  organism  present  in  ten. 
The  diagnosis  of  the  cerebrospinal 
meningitis  may  be  made  by  means 
of  lumbar  puncture,  allowing  the 
spinal  fluid  to  settle,  making  smears 
of  the  sediment  and  staining  by 
means  of  a  blood  stain,  when  the  or- 
ganism may  be  demonstrated,  usually 
inside  of  the  leucocyte,  in  the  form 
of  a  diplococcus  of  a  coffee  bean 
shape  or  as  a  tertracoccus.  It  bears 
a  close  resemblance  to  the  gonococcus. 
It  never  appears  within  the  nucleus 
of  the  polynuclear  leucocyte  and  rare- 
ly within  other  cells.  It  may  be  dis- 
tinguished from  the  other  organisms 
frequently  met  with  in  meningitis 
(pneumococcus,  streptococcus  and 
staphylococcus)  by  its  rapid  decolor- 
ation by  the  Gram  solution.  In  many 
cases  there  are  very  few  diplococci 

.  present  in  the  spinal  fluid,  so  that  a 
failure  to  find  them  -  by  microscopic 
examination  should  not  be  taken  to 
prove  that  the  disease  did  not  exist, 
therefore,  cultures  from  the  fluid 
should  be  made  immediately  upon  its 
withdrawal.  The  organisms  tend  to 
diminish  as  the  disease  advances.  A 
considerable  amount  of  fluid  should 
be  used  for  culture. 

Immunization  of  animals  by  repeated 
inoculation  results  in  the  formation 
of  agglutinins.  A  considerable  per- 
centage of  cultures  are  relatively  in- 
agglutinable.  Strains  that  do  agglu- 
tinate respond  to  the  agglutinins  de- 
veloped in  an  animal  immunized  with 
a  true  strain.  During  recent  years 
attempts  have  been  made  to  treat  the 
disease  by  injections  subcutaneous 
and  intraspinious  of  a  meningococcus 
immune  serum.  Wasserman  in  1907, 
obtained  recoveries  of  32.7%  in  102 
patients  treated  by  the  serum  obtained 
from  horses  immunized  with  pure 
cultures  of  the  meningoccus  and  toxic 
meningococcic  extracts.  Flexner  and 
Jobling   have   more   recently  treated 


BACTERIOLOGY.  197 

the  disease  by  the  use  of  a  similar 
serum  injected  intraspinously,  after 
some  of  the  spinal  fluid  had  been 
withdrawn,  with  excellent  results. 
Hiss  and  Zinsser  claim  to  have  favor- 
ably influenced  the  course  of  the  dis- 
ease by  the  use  of  subcutaneous  in- 
jections of  leucocytic  extract. 

Culturally,  the  organism  grows  between 
25^*  and  40°  C,  best  at  37%°.  Some- 
times it  may  grow  at  23°C.  in  artifi- 
cial media.  While  it  often  lives  for 
weeks,  it  may  die  within  a  few  days 
and  must  therefore  be  transplanted 
to  fresh  material  at  least  every  two 
days.  It  grows  scai*cely  at  all  in 
bouillon.  On  agar,  usually  a  scanty 
growth  appears.  Sometimes  a  few 
colonies  may  grow  vigorously.  Com- 
paratively good  growth  takes  place 
on  Loeffier's  blood  serum,  blood  serum 
or  ascitic  fluid  agar.  Glucose  added 
to  the  media  in  proportion  of  \% 
favors  the  growth.  If  the  organism 
has  been  grown  successfully  for  some 
time,  it  will  produce  a  good  growth 
at  the  end  of  48  hours  on  nutrient 
agar  or  glucose  agar.  The  colonies 
appear  as  a  flat  layer  about  one-eighth 
of  an  inch  in  diameter.  They  are 
greyish  white  in  color,  flnely  granular 
and  nonconfluent  unless  very  close  to- 
gether. On  Loeffier's  blood  serum,  the  ' 
colonies  are  round,  whitish,  shining, 
viscid,  with  smooth  and  sharply  de- 
fined outline.  They  tend  to  become 
confluent  but  do  not  liquefy  the  serum. 

The  organisms  are  readily  killed  by 
heat,  disinfectant,  sunlight  and  dry- 
ing. In  the  dried  state,  a  few  cocci 
may  live  for  one  to  three  days.  After 
the  cultures  have  been  maintained  for 
several  weeks,  by  daily  replanting, 
transplantation  once  a  month  will 
suffice. 

GONOCOCCUS  (BIFIkOCOCCVS 
GONORBKOEA.) 

The  Gonococcus  was  discovered  by 
Neisser  (1879)  in  the  purulent  secre- 
tion of  acute  urethritis  and  vaginitis, 
also  in  the  acute  conjunctivitis  of  the 
new  born.  Bumm  succeeded,  in  1885, 
in  cultivating  this  organism  upon 
human  blood  serum.  He  isolated  the 
organism   in  pure  culture  and  sue- 


198  BACTERIOLOGY. 


ceeded  in  producing  a  disease  by  in- 
oculating- these  cultures  upon  the 
healthy  urethra. 

The  organism  is  usually  seen  in  diplo- 
cocci  form,  flattened  along  the  sur- 
faces, facing  each  other,  which  gives 
it  a  coffee  bean  shape.  Stained  in 
gonorrheal  pus  from  acute  cases,  the 
organisms  are  found  both  intra  and 
extracellularly;  a  great  number  of 
them  are  characteristically  crowded 
within  the  leucocyte.  They  are  never 
found  within  the  nucleus.  The  in- 
tracellular position,  which  is  consid- 
ered diagnostic,  is  not  found  to  any 
great  extent  in  the  secretions  from 
chronic  cases.  It  stains  easily  with 
the  usual  aniline  dyes.  It  is  decolor- 
ized by  Gram,  which  is  of  differential 
value  if  applied  to  the  pus  from  the 
male  urethra.  In  exudates  from  the 
vagina  or  from  the  eye,  the  morph- 
ological characteristics  are  not  so  re- 
liable on  account  of  the  presence  of 
other  Gram  negative  organisms.  In 
the  examination  of  a  chronic  discharge 
for  the  presence  of  the  organism,  it  is 
necessary  to  attempt  cultures  by  rea- 
son of  the  fact  that  a  negative  morph- 
ological examination  cannot  be  re- 
garded as  conclusive. 

A  true  gonnorheal  urethritis  has  not 
been  produced  experimentally  in  an- 
imals. The  infection  occurs  spon- 
taneously In  man.  The  common  seat 
of  infection  is  in  the  male  and  female 
genital  tracts,  in  the  conjunctiva,  and 
it  may  also  produce  a  cystitis,  a  pros- 
tatitis and  stomatitis.  Sometimes  it 
enters  the  blood  stream,  giving  rise  to 
septicemia  and  secondarily  produces 
endocarditis  and  arthritis.  The  or- 
ganism has  been  found  in  a  few  cases 
of  periostitis   and  osteomyelitis. 

Acute  infections  of  the  genito-urinary 
passages  in  man  may  be  followed  by 
a  prolonged*  chronic  infection,  which 
may  remain  quiescent  for  years  and 
be  a  source  of  social  danger. 

In  female  children  particularly,  the  in- 
fection is  not  rare,  and  in  institu- 
tions it  may  travel  from  bed  to  bed 
assuming  epidemic  characters. 

Subcutaneous  and  intraperitoneal  injec- 
tions of  the  organism  into  animals 
would  produce  local  necrosis  and  sup- 
puration, probably  due  to  the  organ- 


BACTERIOLOGY. 


199 


ism's  endotoxin.  This  toxin  has  been 
isolated  by  Nikolaysen  from  the  bac- 
teria by  extracting  with  distilled  , 
water  or  a  solution  of  sodum  hydrate. 
The  toxin  will  resist  a  temperature  of 
a  120°  and  is  fully  as  toxic  for  an- 
imals as  the  living  cultures. 

Christmas  asserts  to  have  demon- 
strated  a   true,   soluble   toxin  which 

,  is  denied  by  Wassermann  and  Niko- 
laysen, who  do  not  believe  that  a 
general  immunity  is  developed  in  in- 
dividuals infected  wit'h  the  organism. 
Christmas  and  Torey  report  success- 
ful immunisation  of  animals,  and 
Torey  has  successfully  treated  human 
cases  by  injections  of  human  serum 
from  immunized  animals.  Bacterins 
have  been  used  with  apparently  real 
benefit  in  inflammations  of  joints  and 
in  very  localized  chronic  infections  of 
the  urethra  and  bladder. 

The  gonococcus  grows  best  at  blood 
temperature.  Its  temperature  range 
is  from  25°  to  40°.  It  may  be  grown 
upon  nutrient  agar  that  has  been 
streaked  with  human  blood.  It  may 
be  grown  on  a  nutrient  agar  contain- 
ing 5%  of  glycerine.  (See  also  media 
for  the  study  of  the  gonococcus). 
After  protracted  cultivation,  the  or- 
ganism will  frequently  grow  on  media 
containing  no  serum.  Some  strains 
will  even  grow  on  plain  nutrient  agar. 

The  cultures  frequently  die  if  kept  at 
room  temperature  for  from  48  to  72 
hours.  In  the  ice-box  they  may  live 
for  several  weeks,  and  on  plain  nu- 
trient agar  they  frequently  live  for 
one  week  at  a  temperature  of  36°  C. 

At  the  end  of  24  hour  cultivations,  a 
delicate  growth  appears,  the  colony 
is  translucent,  finely  granular  with  a 
scalloped  margin,  which  is  sometimes 
scarcely  to  be  differentiated  from  tne 
culture  media.  The  color  is  usually 
greyish  white  with  a  tinge  of  yellow. 
A  streaked  culture  appears  as  a  grey 
white  translucent  growth  with  rather 
thick  edges. 

The  organism  has  but  little  resisting 
powers  against  outside  influences. 
Weak  disinfecting  solutions  kill  it 
readily.  It  does  not  survive  exposure 
to  a  temperature  of  45°  C.  for  six 
hours  or  a  temperature  of  60°  for  . 
thirty  minutes.    Gonorrhea  pus  is  not 


200  BACTERIOLOGY. 


very  resistant  to  desiccation  if  in  thin 
layers,  but  if  smeared  in  thick  layers, 
as  on  linen,  it  has  lived  for  49  days 
and  it  has  also  lived  when  dried  on 
glass  for  29  days. 

MICROCOCCUS  CATABBHAX.IS.  J 

This  organism  is  occasionally  found  iiP 
the  secretions  of  normal  mucous 
membrane,  generally  of  the  respira- 
tory tract,  and  may  be  very  abundant 
in  certain  diseased  conditions  of  the 
mucous  membrane.  At  times  they 
may  produce  catarrhal  inflammations, 
also  pneumonia.  They  occur  in  pairs, 
sometimes  in  fours,  never  in  chains. 
They  are  coffee  bean  in  shape,  slight- 
ly larger  than  the  gonococcus  and 
negative  to  Gram  stain.  According  to 
Ghon  and  PfeifCer  they  are  of  slight 
pathogenic  significance  and  are.  of  im- 
port, aside  from  their  production  of 
catarrhal  inflammation,  only  in  sim- 
ilarity to  the  meningococcus  and  the 
gonococcus.  Certain  cultures  of  the 
micrococcus  catarrhalis  may  prove  as 
pathogenic  for  white  mice,  guinea 
pigs  and  rabbits  as  the  meningococ- 
cus, while  other  cultures  are  less 
pathogenic.  The  range  of  temper- 
ature for  growth  is  from  20*  to  40°; 
optimum,  37^*.  On  nutrient  agar,  the 
growth  appears  as  greyish  white  or 
yellowish  white  circular  colonies  of 
about  the  same  size  as  the  meningo- 
cocci. The  borders  of  the  colony  are 
irregular  and  abrupt.  On  serum  agar, 
the  growth  is  luxuriant.  Gelatin  is 
not  liquefied.  Bouillon  is  clouded  with 
a  frequent  development  of  a  pellicle. 
Milk,  not  changed.  No  gas  production. 
It  is  differentiated  from  the  gonococ- 
cus in  that  it  grows  easily  on  simple 
culture  media  which  is  not  true  of  the 
gonococcus. 

It  is  differentiated  from  the  meningococ- 
cus by  cultural  characteristics  and 
agglutination  reaction.  The  micro- 
coccus catarrhalis  develops  at  tem- 
peratures below  20**  C.  while  the 
meningococcus  does  not  develop  at  a 
temperature  below  25"  C. 

Infections  due  to  the  micrococcus  catar- 
rhalis has  been  successfully  treated 
by  bacterins. 


BACTERIOLOGY.  201 
Pseudp  Meiiingrococcus. 

This  organism  was  described  by  Elser 
and  Huntoon  as  a  diplocoGcus  very 
similar  to  the  meningococcus,  and 
cannot  be  differentiated  from  it  ex- 
cept by  serum  reaction.  It  is  gram 
negative. 

Micrococcus  Fharyngls  Siccus. 

This  organism  was  described  by  von 
Lingelsheim  as  an  organism  similar 
to  the  micrococcus  catarrhalis  from 
which  it  may  be  differentiated  by  fer- 
mentation tests,  and  from  the  men- 
ingococcus and  other  gram  negative 
cocci  by  the  firm  adherence  and  dry- 
ness of  its  colonies. 

Diplococcus  Mucosus. 

This  organism  was  described  by  von 
Lingelsheim.  It  is  similar  to  the 
meningococcus  in  its  colony  formation 
but  more  sticky  and  mucoid.  It  pos- 
sesses a  distinct  capsule,  which  can 
be  demonstrated  by  a  capsule  stain. 
Chromog'eiiic  Gram-ueg'ative  Cocci. 

A  study  of  these  organisms  has  been 
made  by  Elser  and  Huntoon.  They 
produce  a  greenish  yellow  pigment  on 
all  the  ordinary  culture  media.  ,  At 
times  the  pigment  is  absent,  partic- 
ularly when  grown  upon  sugar  free 
culture  miedia,  and  are  to  be  dis- 
tinguished from  the  meningococcus 
only  by  sugar  fermentations  and 
serum  reaction. 

MICBOCOCCUS  MEXiZTENSIS. 
Malta  Fever. 

The  Micrococcus  Melitensis  was  dis- 
covered by  Bruce  in  1887  in  the  spleen 
in  a  case  of  Malta  fever,  and  subse- 
quent investigation  proved  it  to  be  the 
causative  agent  of  Malta  fever.  This 
disease  is  endemic  along  the  shores 
of  the  Mediteranean,  in  South  Africa, 
India,  China,  Japan,  the  West  Indies 
and  the  Philippines.  The  disease  does 
not  seem  to  be  transmitted  from  per- 
son to  person.  The  period  of  incuba- 
tion is  usually  about  6  to  10  days. 
The  ordinary  variety  of  the  fever  is 
intermittent  in  character  and  lasts 
for  a  period  of  from  one  to  three 
weeks  with  intermissions  and  remis- 
sions, and  may  occur  from  time  to 
time  during  a  period  of  many  months, 


202  BACTERIOLOGY. 


accompanied  by  constipation  and  gen- 
eral debilities,  with  various  compli- 
cations, such  as  neuralgia,  arthritis, 
orchitis,  etc,  •  Malignant  cases  have 
been  described,  which  may  be  fatal  in 
a  week  or  ten  days.  -The  mortality  is 
2%  and  at  autopsy  the  spleen  is  found 
to  be  large  and  very  soft.  The  liver 
is  large  and  congested,  both  organs 
having  undergone  parenchymatous  de- 
generation. The  organisms  are  abund- 
ant in  the  blood  and  all  the  organs. 
Animals  eliminate  the  organism  in 
the  urine,  and  milk  of  goats  has  been 
found  to  be  a  prolific  source  of  in- 
fection in  that  the  organisms  are 
passed  with  the  feces  and  so  contam- 
inate the  milk.  Safeguarding  the  milk 
will  largely  eliminate  the  disease. 

The  organism  is  an  oval  coccus,  some- 
times described  as  a  bacillus,  occur- 
Ing  in  pairs,  in  irregular  groups  and 
in  short  chains.  It  is  generally  con- 
sidered non  motile  but  recently  it  has 
been  described  as  motile  and  possess- 
es a  single  flagellum  at  the  ex- 
tremity .  of  the  long  diameter  of  the 
oval  coccus.  It  stains  by  ordinary 
aniline  dyes  and  iis  Gram  negative.  It 

»  grows  at  room  temperature,  best  at 
body  temperature,  either  in  an  acid  or 
alkaline  medium.  The  most  favorable 
media  for  blood  cultures  is  peptone 
broth  to  which  bile  has  been  added. 
On  agar,  after  48  hours,  small,  whitish 
or  yellowish  colonies  appear.  In 
broth,  a  slight  cloudiness  appears 
after  46  days.  The  culure  remains 
alive  for"  several  weeks  or  months. 
The  gelatine  growth  is  very  slow  with 
no  liquefaction. 

Injected  animals  produce  specific  agglu- 
tinins, which  are  of  practical  aid  in 
diagnosis. 

Micrococcus  Zymogreues. 

The  Micrococcus  Zymogenes  was  ob- 
tained by  McCallum  and  Hastings 
from  a  case  of  acute  endocarditis.  It 
has  been  found  in  a  few  other  path- 
ological processes.  The  organisms 
occur  in  pairs  and  sometimes  in  short 
chains.  It  grows  on  agar  and  fer- 
ments lactose  and  glucose.  Gelatine 
is  slowly  liquefied. 


BACTERIOLOGY.  203 


PATHOGENIC  MICBO-OBOANISMS. 

Bacillus  Pyocyaneue. 
(Bacillus  of  Green  azid  of  Bine  Pus.) 

The  blue  and  green  pus  frequently 
found  in  many  suppurating  wounds  is 
due  to  the  action  of  the  bacillus  py- 
ocyaneus.  Gessard,  in  1882,  demon- 
strated this  chromogenic  microorg- 
anism as  a  causative  factor  in  this 
peculiar  type  of  suppuration. 

The  organism  is  usually  found  as  a  short 
straight  rod,  occasionally  slightly 
curved.  The  size  is  subject  to  con- 
siderable variation.  They  are  fre- 
quently united  in  pairs  or  in  chains  of 
4  to  6  elements,  occasionally  growing 
into  long  filaments  and  twisted  spirals. 
Spores  are  not  found.  The  bacillus 
is  actively  motile  and  each  possesses 
a  single  flagellum  placed  at  one  end. 
It  stains  with  the  ordinary  aniline 
dyes  but  not  with  Gram. 

The  bacillus  is  widely  distributed  in 
nature;  it  is  found  on  the  healthy 
skin  of  man,  in  the  feces  of  many 
animals,  in  water  contaminated  by 
animal  or  human  material,  in  pur- 
ulent discharges  and  in  serous  wound 
infections. 

It  is  most  pathogenic  for  guinea  pigs 
and  rabbits.  Subcutaneous  or  in- 
traperitoneal injections  of  1  cc.  of  the 
bouillon  culture  usually  cause  death 
in  from  24  to  36  hours.  When  smaller 
quantities  are  injected  into  subcu- 
taneous tissues,  the  animal  usually 
recovers,  producing  only  a  local  ab- 
scess, and  it  is  subsequently  immune 
against  a  second  inoculation  with  a 
dose  which  would  prove  fatal  to  an 
unprotected  animal. 

In  man,  it  is  found  occasionally  in  con- 
nection with  suppurative  lesions  of 
various  parts  of  the  body;  frequently 
as  a  secondary  infection;  sometimes 
as  the  primary  cause  of  the  infection 
which  does  not  usually  take  place  un- 
less the  individual's  general  condition 
and  resistance  are  abnormally  low. 
Under  such  conditions,  it  may  be  the 
cause  of  chronic  otitis  media.  It  has 
been  cultivated  from  the  stools  of 
children  suffering  from  diarrhea  and 
has  been  found  at  autopsy  distributed 
throughout  the  organs  of  children 
dead  of  gastro-enteritis.  It  has  been 
cultivated  from  the  spleen  at  autopsy 


204  BACTERIOLOGY. 

from  a  case  of  general  sepsis  follow- 
ing mastoid  operations,  Wassermann 
demonstrated  the  bacillus  to  be  an 
etiological  factor  in  an  epidemic  of 
umbilical  infections  in  new  born  chil- 
dren. 

The  organism  is  anaerobic  motile  ba- 
cillus capable  of  growing  anaerobical- 
ly  but  under  this  condition  produces 
no  pigment.  Grows  on  all  artificial 
media  at  room  temperature,  but  best 
at  37°  C.  It  transmits  to  some  of  the 
culture  media  a  bright  green  color  in 
the  presence  of  oxygen.  On  agar,  a 
wrinkled,  moist,  greenish  white  layer 
is  developed.  The  surrounding  me- 
dium is  bright  green,  which  subse- 
quently becomes  darker,  changing  to 
a  blue  green  or  almost  black.  In 
bouillon,  the  growth  appears  as  a 
delicate,  fluorescent  sediment,  chang- 
ing the  color  of  the  bouillon  to  green. 
The  milk  is  coagulated  and  changed 
to  a  yellowish  green  color.  Gelatine 
is  liquefied.  The  liquefaction  in  stab 
cultures  occurs  first  near  the  surface, 
in  the  form  of  a  small  funnel,  and 
extends  downward  and  becomes  strati- 
form,   imparting   a   greenish  yellow 

.  color  to  that  portion  of  the  medium 
which  is  in  contact  with  the  air. 

Two  pigments  are  produced  by  this  or- 
ganism; the  one,  a  fluorescent  green 
common  to  many  bacteria,  is  soluble 
in  water  but  not  in  chloroform;  the 
other,  of  a  blue  color  (pyocynin)  is 
soluble  in  chloroform  and  may  be  ob- 
tained from  pure  solution  in  long 
blue  needles.  This  pigment  dis- 
tinguishes the  bacillus  pyocyaneus 
from  the  other  fluorescing  bacteria. 

Besides  the  ferment  that  causes  the 
liquefaction  of  gelatine,  there  is  one 
which  acts  upon  albumen.  It  is  called 
Pyocyanase,  has  the  power  to  dissolve 
bacteria  and  it  is  believed  to  have 
some  protective  power  when  injected 
Into  animals.  By  reason  of  this  fact, 
it  has  been  used  as  local  treatment  in 
a  number  of  cases  of  diphtheria.  An- 
imal infection  is  followed  by  the  pro- 
duction of  antitoxin  and  antibacteri- 
cidal  substances.  Wassermann  found 
agglutinins  present  in  the  immune 
sera  and  Eisenberg  claims  that  such 
agglutinins  are  active  also  against 
some  of  the  other  fluorescent  bacteria. 


BACTERIOLOGY.  205 


BACIXil^US     PROTEUS  (VUI^GABIS) 

The  Bacillus  Proteus  Vulgaris  was  dis- 
covered along  with  other  species  of 
proteus  by  Houser  (1885),  in  putrefy- 
ing substances.  It  is  one  of  the  most 
widely  distributed  putrefactive  bac- 
teria, and  is  usually  a  harmless  para- 
site when  located  in  the  mucous  mem- 
brane of  the  nasal  cavities  where  it 
only  decomposes  the  secretions  with 
the  production  of  putrefactive  odor. 
Its  pathogenic  powers  are  usually 
slight.  It  is  found  occasionally  in  the 
discharge  from  cases  of  otitis  media 
in  combination  with  other  bacteria. 
Houser  isolated  the  organism  from  a 
case  of  purulent  peritonitis,  from 
purulent  puerperal  endometritis,  and 
from  a  phlegmonous  inflammation  of 
the  hand.  Next  to  bacillus  coli  com- 
munis the  proteus  vulgaris  appears 
most  frequently  concerned  in  the 
etiology  of  pyelonephritis.  In  this 
condition,  together  with  that  of 
cystitis,  the  bacillus  is  frequently 
found  in  pure  culture  or  associated 
with  other  bacteria.  Krogius*  uroba- 
cillus  liquefaciens  septicus  was  prob- 
ably a  variety  of  the  proteus  bacillus. 
Many  epidemics  of  meat  poisoning 
have  also  been  attributed  to  members 
of  the  proteus  family.  Buker,  from 
extended  researches,  concluded  that 
the  proteus  plays  an  important 
part  in  the  morbid  symptoms  which 
characterized  cholera  infantum.  Levi 
obtained  a  pure  culture  from  the 
vomited  material  and  the  stools  in  the 
case  of  a  man  who  shortly  after  died 
of  cholera  morbus.  The  blood  col- 
lected at  the  autopsy  was  sterile. 
Weinsbers  cultivated  a  proteus  ba- 
cillus from  the  putrid  meat  which 
had  caused  acute  gastroenteritis  in  63 
individuals.  Similar  epidemics  have 
been  reported  by  others.  In  some  of 
these  the  bacilli  were  very  toxic  when 
injected  into  animals  but  could  not 
be  recovered  from  the  organs  after 
death.  The  organism  grows  best  at 
temperature  at  or  above  25®  C.  on 
all  media.  It  is  a  facultative  an- 
aerobe. The  bacillus  appears  or  oc- 
curs commonly  as  a  broad,  long  rod 
but  varies  greatly  in  size.  Flexible 


206  BACTERIOLOGY. 


filaments  may  be  formed,  which  are 
sometimes  more  or  less  wavy  or 
twisted  like  braids  of  hair.  It  does 
not  form  spores  and  stains  readily 
with  fuchsin  or  gentian  violet.  In 
broth,  it  produces  a  rapid  clouding 
with  a  pellicle  and  mucoid  sediment 
formation.  In  gelatin,  the  colonies 
are  characteristically  irregular  with 
rapid  liquefaction,  which  is,  however, 
diminished  or  even  inhibited  under 
anaerobic  conditions.  On  agar,  and 
other  solid  media,  the  characteristic 
irregular  colonies  are  produced.  From 
a  central  flat,  greyish  white  poly- 
nucleus  irregular  streamers  grow  out 
over  the  surrounding  medium,  giving 
it  a  stellate  appearance.  On  potato, 
a  dirty  yellowish  growth  appears. 
Milk  is  coagulated  with  an  acid  reac- 
tion at  first,  later  the  casein  is  redis- 
solved.  In  peptone  solutions,  endol 
and  phenol  is  produced.  It  grows  well 
in  urine  and  decomposes  urea  into 
carbonate  of  ammonia. 

BACIIiZknS  MAI^IiEZ. 

Glander  Bacillus. 

TJie  Bacillus  of  Glanders  was  first  ob 
tained  in  pure  culture  by  Loeffler  and 
Schutz  in  1882.  It  causes  an  infec- 
tious disease  called  glanders,  which  is 
prevalent  chiefly  among  horses,  but  is 
occasionally  transmitted  to  other 
domestic  animals  and  man. 

The  organism  is  a  rather  small  rod  with 
rounded  ends,  usually  straight,  but 
may  be  slightly  curved.  Separate  in- 
dividuals in  the  same  culture  vary 
greatly  in  size  and  this  is  a  charac- 
teristic of  the  organism.  In  old  cul- 
tures, involutions  appear  as  short, 
vacuolated  almost  coccoid  individuals. 
It  is  stained  easily  with  methylene 
blue,  showing  irregularity  in  its 
staining  qualities;  the  deeply  stained 
areas  alternate  with  areas  that  are 
faintly  stained  or  entirely  unstained. 
This  staining  irregularity  is  charac- 
teristic. The  organism  is  non  motile 
and  does  not  form  spores.  It  is  decol- 
orized by  Gram's. 

Infection  with  the  glander  bacillus  oc- 
curs spontaneously  most  frequently  in 
horses.  It  may  occur  in  asses,  cats 
and  more  rarely  in  dogs.  The  disease, 
in    some    cases,    infects    man,    if  in 


BACTERIOLOGY':  207 


habitual,  contact  with  horses.  Cattle, 
dogs,  rats  and  birds  are  immune.  Ex- 
perimental inoculations  have  been  suc- 
cessful in  guinea  pigs  and  rabbits. 

The  infection  takes  place  through  the 
mucosa  of  the  mouth  and  nasal  pass- 
ages and  occasionally  through  the  di- 
gestive tract  in  horses.  It  is  believed 
that  injury  to  the  skin  or  mucosa  is 
necessary  for  the  entrance  and  the 
development  of  the  bacilli. 

In  horses,  the  disease  occurs  in  an 
acute  or  chronic  form,  depending  upon 
the  susceptibility  of  the  subject  or  the 
relative  virulence  of  the  organism. 
The  acute  form  of  the  disease  is 
usually  limited  to  the  nasal  mucosa 
and  the  upper  respiratory  passages. 
It  begins  with  fever  and  prostration 
after  2  or  3  days;  there  is  at  first  a 
^serous  nasal  discharge  whiph  later  be- 
comes seropurulent;  coincident  with 
this,  there  is  ulceration  of  the  nasal 
mucosa  and  swelling  of  the  neighbor- 
ing lymphatic  glands,  which  may 
break  down  and  form  pus  discharging 
sinuses  and  ulcers.  The  lungs  now 
become  involved  and  death  follows 
within  4  to  6  weeks.  The  chronic  type 
is  accompanied  by  multiple  swellings 
of  the  skin  and  general  lymphatic  en- 
largement, and  in  this  form  is  spoken 
of  as  "farcy."  In  this  type,  the  onset 
is  more  gradual,  together  with  the 
nasal  inflammation.  The  swelling  of 
the  skin  in  some  cases  shows  a  tend- 
ency to  break  down  and  ulcerate.  The 
disease  may  last  for  several  years 
and  may  occasionally  end  in  a  cure. 
This  is  by  far  the  most  frequent  type 
of  the  disease  in  horses.  When  the 
disease  occurs  in  man,  it  is  quite  like 
that  of  the  horse  except  that  the 
point  of  origin  is  more  frequently  in 
the  skin  than  in  the  nasal  mucosa. 
The  onset  of  the  disease  is  violent, 
with  fever  and  systemic  symptoms.  A 
nodule  appears  at  the  point  of  infec- 
tion surrounded  by  lymphangitis  and 
swelling.  Occasionally  a  papular 
eruption  occurs,  which  may  become 
pustular  and  clinically  may  simulates 
small  pox.  Death  usually  follows  in 
8  to  10  days.  The  chronic  form  in 
man  is  much  like  that  in  the  horse, 
^but  is  more  often  fatal. 


208  B  ACTERIOLOGY. 

The  diagnosis  of  glanders  may  be  made 
by  isolating  and  indentifying  the  or- 
ganism  from  the  center  of  the  glander 
Tiodule,  the  nasal  secretions  and  occa- 
sionally from  the  blood.  In  the  ma- 
jority of  cases,  however,  isolation  is 
difficult  and  animal  inoculation  be- 
comes a  necessity.  Intraperitoneal 
inoculation  with  material  containing 
the  bacillus  is  made  into  male  guinea 
pigs,  which  leads  within  two  or  three 
days  to  tumefaction  and  inflammation 
of  the  testicles.  This  method  is  spoken 
of  as  "Strauss  Test,"  which  should  be 
reinforced  by  cultures  of  the  testic- 
ular pus,  the  spleen  and  the  peritoneal 
exudate  of  the  animal  on  potato. 

The  organism  is  aerobic  but  growths 
may  also  take  place  under  an- 
aerobic conditions.  The  temperature 
range  is  22°  to  43'  C,  optimum  37 
C.  It  grows  easily  on  all  culture 
media,  whether  neutral  or  slightly 
alkaline  or  slightly  acid  in  reaction. 
Glycerine  or  small  quantities  of  glu- 
cose added  to  the  media  favors  its 
cultivation.  On  agar,  the  colonies  ap- 
pear after  24  hours  as  yellowish 
white,  first  transparent,  later  opaque 
•spots,  with  even  border.  Old  cultures 
become  more  yellow.  On  gelatin  the 
the  growth  is  slow,  of  a  greyish  white 
color,  with  no  liquefaction.  In  broth, 
there  is  at  first  (clouding  later)  a 
heavy,  slimy  sediment  with  pellicle 
formation.  Broth  later  assumes  a 
brown  color.  Milk  is  coagulated. 
Litmus  milk  indicates  acid.  On  potato 
media,  which  is  not  too  acid,  an 
abundant  growth  appears  within  48 
hours,  completely  covering  the  surface 
as  a  yellowish,  transparent,  slimy 
mass,  which  grows  darker  until  it  be- 
comes a  deep  reddish  brown.  This 
growth  is  considered  diagnostic,  al- 
though the  potato  growth  of  the  ba- 
cillus pyocyaneous  is  very  similar. 

The  culture  will,  if  kept  cool  and  in  the 
dark  in  sealed  tubes,  live  for  months 
and  years.  Sunlight,  if  strong,  will 
kill  it  within  24  hours.  Heat  will 
kill  it  if  exposed  for  2  hours  to  60"  C; 
one  hour  if  75°  C.  Its  resistance  to 
chemical  disinfections  as  well  as  dry- 
ing is  not  very  high. 

The  toxin  (Mallein)  belongs  to  the  class 
of  endotoxins  and  is  obtained  by  ex- 


BACTERIOLOGY.  209 

traction  of  dead  bacilli  (see  Mallein 
under  vaccines).  It  differs  from  other 
bacterial  poisons  in  its  extreme  re- 
sistance to  temperatures  of  a  120**  C. 
and  prolonged  storage.  It  is  not  a 
powerful  poison  to  healthy  animals,  as 
,  considerable  doses  can  be  given  with- 
out producing  death.  It  is  used  for 
diagnostic  purposes.  The  injection  of 
the  Malein  may  cause  reactions  in  the 
presence  of  other  diseases  than  gland- 
ers, such  as  bronchitis,  periostitis, 
etc.,  and  is  therefore  not  so  valuable 
specifically  as  tuberculin  for  diag- 
nosis. 

Recovery  from  glanders  will  not  pro- 
duce immunity.  Agglutinins  are  form- 
ed in  the  serum  of  subjects  suffering 
from  the  disease,  and  may  be  used 
for  diagnostic  purposes  if  used  in 
dilutions  of  at  least  on6  to  five 
hundred. 

BACIXiXinS  AITTKBACZS. 

The  bacillus  of  anthrax  was  first  ob- 
served in  the  blood  of  infected  an- 
imals by  Pollender  in  1849.  Experi- 
mental infection  in  animals  with  the 
blood  containing  the  bacilli  was  made 
by  Davaine  in  1863. 

The  bacillus  causes  an  acute  infectious 
disease.  Is  very  prevalent  among  an- 
imals, particularly,  sheep  and  cattle. 
The  infection  not  infrequently  occurs 
in  horses,  hogs  and  goats.  It  is  the 
most  wide  spread  of  all  infectious 
diseases.  It  is  more  common  in 
Europe  and  Asia  than  in  America.  Its 
ravages  among  the  cattle  in  Russia 
and  Siberia,  the  sheep  in  France, 
Hungary,  Germany,  Persia  and  India 
have  been  more  severe  than  those  of 
any  other  animal  plague.  Local  epi- 
demics have  occured  in  England  and 
it  is  there  called  Splenic  Fever. 

The  disease  also  occurs  in  man,  particu- 
larly among  stablemen,  shepherds, 
tanners,  butchers  and  those  who  work 
in  wool  and  hair.  Two  forms  of  the 
disease  have'  been  described;  the  ex- 
ternal anthrax,  or  malignant  pustules, 
and  the  internal  anthrax  of  which 
there  are  intestinal  and  pulmonary 
forms,  the  so-called  wool-sorter's  dis- 
ease. The  bacillus  is  a  *non  motile 
straight  rod.  In  the  blood  of  animals, 
they  are  usually  single  or  in  pairs. 
Grown  on  artificial  media,  they  form 


210  BACTERIOLOGY. 


long  threads.  The  end  of  the  indi- 
vidual bacillus  is  square.  In  the 
threads,  the  corners  of  the  bacillus  are 
so  sharp  that  the  ends  in  contact  in 
a  chain  often  touch  each  other  only 
at  the  corner,  leaving  an  oval  chink 
between  the  ends  of  the  organism.  On. 
artificial  media,  the  organism  forms 
oval  spores,  centrally  located,  which 
are  not  found  in  the  blood  of  animals.  *. 
The  organisms  are  easily  stained  by 
the  usual  aniline  dyes  and  are  gram 
positive.  Especially  stained  organisms 
from  the  animal  tissues  or  the  blood 
occasionally  shows  a  capsule.  This 
has  never  been  demonstrated  in  cul- 
tures on  ordinary  media. 

The  anthrax  bacillus  is  pathogenic  for 
cattle,  sheep,  guinea  pigs  rats  and 
mice,  their  degree  of  susceptibility 
varying  greatly,  even  among  different 
members  of  the  same  species,  as 
shown  by  the  high  resistance  of 
Algerian  sheep  and  the  high  sus- 
ceptibility of  the  European  variety. 
Dogs,  hogs,  cats,  birds  and  cold 
blooded  animals  are  relatively  im- 
mune. The  organism  is  definitely 
.pathogenic  to  man  though  less  so  than 
for  cattle,  etc. 

Separate  races  of  the  organism  may 
vary  much  in  their  virulence.  A  single 
strain  may  remain  constant  as  to  viru- 
lence, if  preserved,  dried  upon  threads 
or  kept  in  sealed  tubes  in  dark  places. 
The  virulence  of  the  organism  may 
be  reduced  by  the  various  attenuat- 
ing procedures,  which  is  of  import- 
ance in  prophylactic  immunization. 
Experimental  inoculation  subcutane- 
ously  is  followed  at  first  by  no  symp- 
toms, and  some  animals  appear  per- 
fectly well  until  a  few  hours  or  less^ 
before  death.  The  duration  of  the  ^ 
disease  depends  upon  the  resistance 
of  the  infected  subject.  The  quantity 
of  the  infectious  material  injected 
has  little  bearing  on  the  outcome,  as 
a  single  bacillus  is  frequently  suffi- 
cient to  bring  about  a  fatal  result. 
The  bacilli  are  not  found  in  the  blood 
until  immediately  before  death.  They 
are,  however,  conveyed  from  the  point 
of  inoculation  by  the  blood  and 
lymph  streams  to  all  the  organs,  as 
has  been  demonstrated  when  the  tail 
or    ear    of   animals   was    inoculated,  1 


BACTERIOLOGY.  211 


without  preventing  a  fatal  infection. 
The  bacilli  do  not,  as  a  rule,  multiply 
in  the  blood,  at  least,  not  at  first. 
They  may  proliferate  at  the  point  of 
inoculation  and  probably  in  the  or- 
g-ans  and  when  the  resistance  of  the 
animal  is  overcome.  They  invade  the 
circulation  and  multiply  within  it. 
Autopsy  upon  suoh  animals  shows  at 
the  point  of  inoculation,  edematous, 
hemorrhagic  infiltration.  The  spleen 
is  congested  and  enlarged.  The  kid- 
neys are  congested  and  there  may  be 
hemorrhagic  spots  upon  the  mucous 
membrane.  Death  brought  about  by 
the  anthrax  bacillus  is  probably  due 
in  a  large  extent  to  obstruction  of 
the  capillaries,  although  a  true  toxin 
has  never  been  demonstrated,  the 
toxic  clinical  picture  of  the  disease 
presented  in  some  animals  and  in 
man  precludes  the  possibility  that 
such  poisons  do  not  exist,  though 
neither  the  culture  filtrates  nor  the 
dead  bacilli  have  any  noticeable  toXic 
effect  upon  test  animals  and  exert  no 
immunizing  action. 
Infection  of  animals  takes  place  by 
way  of  the  alimentary  canal.  The 
sjpores  of  the  bacteria  resist  the  g^r^ 
trie  juice  and  develop  into  the  vegeta- 
tive form  in  the  intestines,  where 
they  increase  and  invade  the  system. 
Subcutaneous  infections  may  occur 
when  there  are  small  punctures  and 
abrasions  about  the  mouth.  When 
Infection  takes  place  upon  a  visible 
part,  there  is  formed  a  diffused  local 
swelling,  somewhat  like  a  large  car- 
buncle, the  center  of  which  is  marked 
by  a  black,  necrotic  slough,  or  may 
contain  a  pustular  depression.  In- 
fection by  way  of  inhalation  is  rare 
among  animals.  The  disease  in  in- 
fected cattle  and  sheep  is  very  acute 
and  kills  within  one  or  two  days. 
There  is  about  80%  mortality.  In 
man  infection  generally  takes  place 
through  a  small  cutaneous  abrasion. 
It  may  also  occur  by  inhalation  and 
through  the  alimentary  tract.  The 
cutaneous  infection  occurs  usually 
through  an  abrasion  of  the  skin  in 
men  who  handle  live  stock,  in 
butchers  and  tanners  of  hides.  The 
primary  lesion,  appearing  at  the  sight 
of  inoculation  within  12  to  24  hours, 


212  BACTERIOLOGY. 

resembles  an  ordinary  small  furuncle, 
with  a  central  vesicle  filled  with  a 
sero  sanguineous  and  later  a  sero 
purulent  fluid.  This  changes  in  the 
center  to  a  black,  necrotic  mass 
which  is  surrounded  by  an  edematous 
areola.  If  early  and  prompt  excision 
Is  made  of  the  mass,  the  patient  re- 
covers; if  not,  local  gangrene  and 
general  systemic  infection  may  occur 
and  lead  to  death  within  5  or  6  days. 
The  pulmonary  Infection  (wool-sorter's 
disease),  rare  In  this  country,  occurs 
in  persons  who  handle  raw  wool, 
hides  or  horse  hair,  by  their  inhaling 
or  by  their  swallowing  the  spores. 
The  disease  has  manifested  itself  as 
a  violent,  irregular  pneumonia,  which, 
In  the  majority  of  cases,  leads  to 
death. 

Infection  through  the  alimentary  canal, 
rare  in  man,  usually  takes  place 
through  the  ingestion  of  uncooked, 
infected  meat,  the  initial  lesion  locat- 
ing in  the  small  intestine,  producing 
violent  enteritis,  with  bloody  stools 
and  great  prostration,  which  results 
in  death. 

The  anthrax  bacillus  grows  very  lux- 
uriantly under  aerobic  conditions, 
while  it  develops  slowly  and  tersely 
under  anaerobic  conditions.  Its  op- 
timum temperature  is  37^'  C.  but 
will  grow  at  temperature  as  low 
as  15°  and  as  high  at  45*.  It  may  be 
cultivated  upon  all  the  ordinary 
media;  a  slightly  alkaline  or  neutral 
media  seems  to  be  the  optimum  reac- 
tion. On  agar  plates,  vigorous  colonies 
appear  in  12  to  24  hours.  They  are 
irregular  in  outline,  wrinkled,  and  if 
examined  under  the  microscope,  they 
seem  to  be  made  up  of  hair-like 
tangle  of  thread  spreading  in  wavy 
layers  from  a  more  compact  central 
knot.  On  gelatin  plates,  they  appear 
within  24  to  48  hours  as  opaque  pin 
head  size  white  disks.  As  the  colony 
increases  in  size,  their  outline  be- 
comes less  regular,  and  under  the 
microscope  Is  similar  to  that  of  the 
agar  plates.  Liquefaction  takes  place 
in  about  three  or  four  days.  In  the 
gelatin  stab  there  is  at  first  a  thin, 
white  line  along  the  puncture.  This 
growth  continues  into  the  formation 
of  thin  filaments,  which  diverge  from 


BACTERIOLOGY.  213 

the  stab  and  take  on  an  appearance 
not  unlike  a  small,  inverted  "Christ- 
mas tree."  Liquefaction  begins  at 
the  top.  In  broth  there  is  at  first  a 
rapid  growth  with  uneven  clouding 
and  a  pellicle  formation,  later  a 
slimy  mass  somewhat  like  a  cotton 
fluff  appears.  On  potato,  there  is  a 
rapid,  white,  dry  growth.  Milk  is 
slowly  acidified  and  coagulated. 

On  account  of  the  spores,  the  anthrax 
is  extremely  resistant  to  chemical 
and  physical  agents.  The  vegetative 
form  is  killed  by  an  exposure  to  a 
temperature  of  54 C.  for  ten  minutes. 
The  spores  in  a  dry  state  will  live 
for  many  years.  The  exposure  of  the 
spore  to  dry  heat  at  140°  C,  for 
three  hours,  is  necessary  to  kill.  Live 
steam  at  100°  C.kills  them  in  from  5  to 
10  minutes.  Low  temperature  does 
not  seem  to  have  a  great  deal  of  ef- 
fect on  them.  The  spore's  resistance 
to  chemicals  varies  with  different 
strains.  Direct  sunlight  will  k411  the 
spore  within  6  to  12  hours. 

Active  immunization  of  small  labora- 
tory animals  is  very  difficult  but  can 
be  accomplished  wih  extremely  at- 
tenuated cultures. 

Passive  immunity  by  means  of  serum, 
of  actively  immunized  sheep  has  been 
produced  and  practically  applied  by 
Sobernheim.  The  injection  of  such 
serum  has  been  found  to  protect  an- 
imals from  anthrax  and  to  confer  an 
immunity  which  lasts  often  as  long 
as  two  months. 

No  specific  nor  bactericidal  nor  bac- 
terialitic  properties  have  been  dem- 
onstrated in  the  immune  serum.  Ag- 
glutinins have  not  been  satisfactorily 
demonstrated. 

ANTHBAX-IiIKE  BACIIiU. 

In  nearly  all  laboratories  there  are 
strains  of  true  anthrax  bacilli  which 
have  become  so  attentuated  that  they 
are  practically  non  pathogenic.  They 
do  not,  however,  differ  from  the  viru- 
lent organisms  in  their  culture  or 
morphological  characteristics.  There 
are  likewise  in  the  laboratory  certain 
non  virulent  bacteria  which  do  not 
resemble  the  anthrax  bacillus  cultur- 
ally but  do  so  morphologically  (see 
below). 


214  BACTERIOLOGY. 


Bacillus  Anthracoides.  This  is  a  non- 
pathogenic, gram  positive  organism, 
indistinguishable  from  the  bacillus 
anthracis,  except  morphologically  the 
ends  are  more  rounded  and  culturally 
the  growth  is  more  rapid,  together 
with  a  more  rapid  liquefaction  of 
gelatin. 

Bacillus  Badicosus.  This  is  a  non- 
pathogenic organism  cultivated  from 
city  water  supplies.  Morphologically, 
it  is  somewhat  larger  than  the  an- 
thrax bacillus  and  the  individual  ba- 
cilli are  more  irregular  in  size.  Cul- 
turally, the  growth  is  most  active  at 
room  temperature,  with  very  rapid 
liquefaction  of  gelatin. 

Bacillus  SufetUis.  (Hay  Bacillus).  This 
is  a  practically  non-pathogenic  Gram 
positive  organism  found  in  brackish 
waters  and  infusions  of  vegetable 
matter,  and  occasionally  as  a  saphro- 
phyte  or  secondary  invader  in  chronic 
suppurative  lesions,  as  in  old  sinuses 
an^  infected  wounds.  It  is  not  very 
closely  related  to  the  anthrax  bacillus. 
It  occurs  as  straight  rods  and  is 
actively  motile  in  young  cultures  in 
which  the  bacilli  appear  singly  or  in 

»  pairs.  In  older  cultures,  chains  are 
formed  and  the  bacilli  become  mo- 
tionless. Spores  are  formed,  usually 
slightly  nearer  one  pole  than  the 
other.  On  gelatin  and  agar  the  ba- 
cilla  grow  as  a  dry,  corrugated  pelli- 
cle. Gelatin  is  liquefied.  Micro- 
scopically the  colonies  are  irregularly 
round  with  fringed  edges  and  made 
up  of  interlacing  threads. 

BACIZiI^US  DIPHTHERIA. 
(Xlebs-Zioeffler  Bacillus) 

The  -  Bacillus  of  Diphtheria  was  dis- 
covered by  Klebs  in  1883,  having  ob- 
served the  bacillus  morphologically 
from  the  pseudo  membranes  of  diph- 
theritic throats.  An  organism  was 
isolated  and  cultivated  by  Loeffler  in 
1884,  which  corresponded  to  the 
morphological  characters  of  the  ba- 
cillus discovered  by  Klebs.  He  in- 
oculated the  organisms  upon  the  in- 
jured mucous  membrane  of  animals 
and  produced  lesions  which  resembled 
the  false  membranes  of  the  disease  in 
human  individuals.  Loeffler  was, 
however,  very  conservative  in  nis  de- 


BACTERIOLOGY.  215 


scription  of  this  organism  as  a 
causative  agent  of  diphtheria  by  rea- 
son of  his  failure  to  find  the  organ- 
ism in  all  cases  examined,  and  his 
finding  the  organism  in  the  throats 
of  a  liealthy  individual,  together  with 
his  inability  to  explain  the  systemic 
manifestations  of  the  infection.  All 
existing  doubt  as  to  the  etiology  of 
the  disease  was  overcome  by  Loeffler's 
further  studies  together  with  the 
publication  of  articles,  on  the  nature 
of  the  toxin  produced  by  the  diph- 
theria bacillus,  by  Roux  and  Yersin 
m  1888. 

The  bacillus  of  diphtheria  is  subject  to 
a  number  of  morphological  variations 
which  depend  to  a  certain  extent  upon 
the  age  of  the  culture  and  upon  the  con- 
stitution of  the  medium  of  which  it  is 
grown.  These  factors  do  not,  however, 
control  the  appearance  of  the  organism 
with  any  degree  of  regularity,  in  as 
much  as  all  of  the  variations  may  be 
observed  in  the  same  growth.  The 
difference  in  its  morphology  probably 
represents  stages  in  the  growth  and 
degeneration  of  the  individual  organ- 
ism. Certain  characteristics  in  the 
morphology  of  the  organism  facili- 
tate its  recognition.  They  appear  as 
slender,  straight  or  slightly  curved 
rods.  Their  thickness  throughout 
their  length  Is  rarely  uniform.  They 
may  show  club  shaped  thickenings  at 
one  or  both  ends.  Sometimes  they 
are  more  thick  at  the  center  and 
taper  towards  the  ends.  If  they  are 
thickened  at  one  end,  they  take  on  a 
slender  wedge-shaped  form,  and  are 
usually  straight,  of  smaller  size  than 
the  others  mentioned  and  stain  uni- 
formly. This  type  has  been  re#Brred 
to  by  Beck  as  "ground  type"  and  be- 
lieved by  him  to  be  young  individual. 
Branched  forms  have  also  been  noted, 
which  are  probably  abnormal  or  in- 
volution forms.  The  organisms  stain 
readily  with  watery  aniline  dyes.  An 
irregularity  of  staining  is  a  charac- 
teristic of  diagnostic  value  and  is 
best  obtained  by  the  use  of  Loefller's 
alkaline  methylene  blue,  which,  if  ap- 
plied for  from  5  to  10  minutes,  will 
cause  the  bacilli  to  be  traversed  by 
stained  and  unstained  bands,  which 
give  to  the   organisms  a  striped  or 


216  BACTERIOLOGY. 

beaded  appearance.  If  the  organisms 
are  long,  they  may  take  on  the  ap- 
pearance of  short  streptococci;  others 
may  appear  granular.  The  bacilli  of 
abput  18  hours'  culture  may  show 
stained  oval  bodies,  most  frequently 
situated  at  the  end,  and  are  spoken 
of  as  polar  or  Babes-Ernst  bodies. 
Special  stains  for  these  bodies  have 
been  brought  out  by  Neisser  and 
others,  who  claim  for  them  differen- 
tial value  in  distinguishing  the  diph- 
theria bacillus  from  other  morph- 
ologically like  organisms.  These  polar 
bodies  are  probably  chromatic  gran- 
ules. The  organism  is  stained  by 
Gram,  but  care  must  be  taken  in 
timing  the  stain.  Carelessness  may 
lead  to  irregular  results. 

The  bacillus  of  diphtheria  causes  a 
specific  local  action  upon  mucous 
membranes,  the  so-called  pseudo 
membrane.  The  disease  depends  in 
part  uiibn  the  mechanical  surface  of 
this  membrane,  and  in  part  upon  the 
toxins  which  are  produced  by  the 
organism.  The  most  frequent  sites 
of  diphtheria  are  the  mucous  mem- 
branes of  the  throat,  larynx  and  nose. 
»  Occasionally  they  have  been  found  in 
the  ear,  upon  thfe  mucous  membrane 
of  the  stomach  and  vulva  and  upon 
the  conjunctiva  and  skin.  The  or- 
ganism may  extend  from  the  larynx 
and  cause  a  diphtheritic  broncho- 
pneumonia. Although  the  organism 
has  been  isolated  after  death  from  the 
spleen  and  liver,  a  true  diphtheritic 
speticemia  is  not  probable. 

The  organism  is  very  pathogenic  to 
dogs,  cats,  fowls,  rabbits  and  guinea 
pigs.  Rats  and  mice  will  resist  it, 
if*  administered  in  extremely  large 
doses.  Membranes  analogous  to  those 
found  in  man,  have  been  prod?uced  in 
susceptible  animals,  but  only  when 
mechanical  injury  to  the  mucosa  has 
preceded  the  inoculation  with  the 
bacillus.  If  small  quantities  (one- 
half  to  1  cc.)  of  a  broth  culture  are 
Injected  subcutaneously  into  a  guinea 
pig,  symptoms  appear  within  6  to  8 
hours  which  are  followed  by  death 
within  36  to  72  hours.  At  autopsy, 
a  serosanguineous  exudate  will  be 
found  at  the  point  of  inoculation. 
The    lymph    glands    are  edematous. 


BACTERIOLOGY.  217 

The  kidney,  liver,  speen  and  lungs 
are  congested.  There  may  be  exudates 
in  the  pleural  and  peritoneal  cavities. 
A  severe  congestion  of  the  suprarenal 
bodies  is  characteristic  and  almost 
pathognomonic. 

Agglutinins  for  the  diphtheria  bacillus 
have  been  developed  in  an  amount  to 
act  in  one  to  five  thousand  dilutions 
of  the  serum  by  the  injections  of  the 
bodies  of  the  organism  into  animals. 
The  serum  of  convalescent  patients 
has  ordinarily  but  little  agglutinating 
power.  The  test  is  not  used  in  diag- 
nosis. Antitoxins  have  been  prepared 
and  used  in  treatment  with  very 
beneficial  results  (see  diphtheria  anti- 
toxin). Active  immunization  has  been 
recommended  by  Theobald  Smith  by 
the  use  of  mixtures  of  toxin  and  anti- 
toxin, in  this  way  producing  an  im- 
munity. There  are  a  great  number 
of  objections  to  this  method  of  im- 
munization. 

The  organism  is  an  aerobe,  but  will 
grow  under  anaerobic  conditions  in 
the  presence  of  carbohydrate.  The 
temperature  range  is  19"  to  42°  C, 
optimum  37%**  C.  Any  temperature 
above  this  impedes  the  development 
of  the  toxin.  The  organism  is  iso- 
lated from  mixed  cultures  very  read- 
ily. Cultures  are  taken  from  throats 
and  placed  upon  Loeffler's  blood  se- 
rum, upon  which  they  are  permitted 
to  grow  at  371/^*  C.  for  from  18  to  24 
hours.  An  emulsion  is  now  made  from 
the  growth  with  about  5  cc.  of  bouil- 
lon; two  or  three  loopfuls  of  this  emul- 
sion is  streaked  over  the  surface  of 
a  sugared  agar,  incubated  for  24 
hours  and  the  characteristic  colonies 
transferred  to  Loeffler's  media. 

The  diphtheria  bacillus  grows  readily 
on  most  of  the  rich  laboratory  media. 
The  most  favorably  reaction  for  its 
growth  is  probably  about  alka- 
linity. Loeffler's  media  is  the  most 
widely  used  media  for  the  cultivation 
of  the  organism.  Swabs  from  sus- 
pected throats  are  smeared  over  the 
surface  of  Loeffier  media  and  incu- 
bated at  37%**  C.  At  the  end  of  12 
to  24  hours  minute,  greyish  white, 
bristling  colonies  of  the  diphtheria 
bacillus  are  developed.  These  enlarge 
and  grow  to  such  an  extent  that  they 


218  BACTERIOLOGY. 


outstrijp  the  accompanying  micro- 
org-anisms.  This  method  is  of  value 
for  diagnostic  purposes.  On  agar,  the 
colonies  appear  within  24  to  36  hours 
as  small,  translucent,  greyish  spots, 
quite  characteristic  and  easily  recog- 
nized. The  colonies  become  irreg- 
ularly round  or  oval,  with  a  dark 
nucleus  like  center,  fringed  by  a  loose, 
coarse,  granular  disk.  The  edges  of 
the  colony  are  irregular.  The  addi- 
tion of  1%  dextrose,  2%  nutrose, 
6%  glycerine  renders  the  agar  favor- 
able for  rapid  growth  but  makes  it 
a  very  poor  media  for  the  preserva- 
tion of  the  culture.  A  meat  infusion 
gelatin  is  a  favorable  media  but  be- 
cause of  the  low  temperature  at  which 
the  media  must  be  kept,  the  growth  is 
very  slow.  Gelatin  is  not  liquefied. 
The  organism  grows  readily  in  milk 
with  no  coagulation.  Endol  is  not  pro- 
duced in  peptone  solutions. 
The  organism  has  a  thermal  death  point 
of  58°  C.  It  is  killed  if  exposed  to  a 
boiling  heat  for  one  minute.  Low 
temperatures  are  borne  readily.  Desic- 
cation and  exposure  to  light  are  not 
as  fatal  to  it  as  to  most  other  patho- 
,  genie  organisms.  Chemical  disin- 
fectants will  kill  the  organisms 
readily. 

PSEUDO     DIPHTHERIA  BACIZiZkUS. 

(Bacillus  Hoffmaimi). 

The  Pseudo  diphtheria  Bacillus  was 
described  by  Hoffmann  in  1888,  who 
cultivated  the  organism  from  the 
throats  of  normal  individuals,  and  in 
several  instances  from  the  throats 
of  diphtheritic  persons.  The  organ- 
ism resembles  the  diphtheria  bacillus 
but  differs  from  it  in  its  non-patho- 
genicity  to  guinea  pigs.  It  was  at 
first  regarded  as  an  attentuated  form 
of  diphtheria  bacillus,  but  further 
study  showed  it  to  be. unquestionably  a 
separate  species.  It  is  a  non-motile 
bacillus,  shorter  and  broader  than  the 
bacillus  of  diphtheria.  It  is  usually 
straight  and  may  be  slightly  clubbed 
at  one  end.  Stained  with  Loeffler's 
methylene  blue,  it  may  show  un- 
stained bands,  but  unlike  the  diph- 
theria bacillus,  these  bands  rarely 
number    more    than    one,    and  never 


BACTERIOLOGY. 


219 


more  than  two.  No  polar  bodies  have 
been  demonstrated  by  special  stains. 
Distinguished  culturally  from  the  diph- 
theria bacillus,  it  grows  more  lux- 
uriantly upon  simple  media.  On  agar 
plates,  the  colonies  are  larger,  less 
transparent  and  more  white.  In  liquid 
media,  there  is  clouding  and  less 
tendency  to  pellicle  formation.  It 
does  not  form  acid  upon  the  various 
sugared  media.  Animals  immunized 
with  it  do  not  possess  increased  re- 
sistance to  the  diphtheria  bacillus.  It 
is  entirely  inocuous  to  ordinary 
laboratory  animals. 

BACZI.I.US  XEROSIS. 

The  Bacillus  Xerosis,  almost  indentical 
with  the  diphtheria  bacillus,  was  dis- 
covered by  'Kutschert  and  Neisser 
from  the  eyes  of  patients  suffering 
from  a  ,  chronic  conjunctivitis  called 
Xerosis.  They  believed  it  to  be  the 
etiological  factor  in  this  disease,  but 
the  fact  that  it  has  been  frequently 
isolated  from  normal  eyes,  precluded 
it  as  a  causative  factor.  It  is  prob- 
ably a  harmless  parasite,  found  more 
often  in  the  slightly  inflamed  than 
in  the  normal  conjunctiva, 

No  absolute  differentiation  morpholog- 
ically can  be  made  between  the  diph- 
theria bacillus  and  the  bacillus 
xerosis.  Polar  bodies  have  occasion- 
ally been  seen.  Its  growth  on  Loef- 
fler's  blood  serum  agar,  glycerine  agar 
and  in  broth  is  probably  more  delicate 
but  very  similar  to  that  of  bacillus 
diphtheria.  It  is  not  easily  cultivated 
upon  ordinary  meat  extract  media. 
It  will  not  grow  on  gelatin  at  room 
temperature  and  on  glycerine  or  glu- 
cose agar;  the  colonies  are  micro- 
scopically identical  with  those  of 
diphtheria.  It  differs,  however,  from 
the  bacillus  of  diphtheria  in  produc- 
ing acid  from  saccharose  but  not  from 
dextrin.  It  is  non  pathogenic  to  an- 
imals and  does  not  form  a  toxin. 

BACIZiI^US  INFI^UENZA. 
(Pfelffer  Bacillus). 

Epidemics  of  influenza  can  be  traced 
back  to  the  fifteenth  century.  The  last 
serious  epidemic  occurred  in  the  years 
1889  to  1890.  Beginning  in  the  East, 
traveled  through  Russia,  became  pan- 


220  BACTERIOLOGY. 


demic  in  Europe,  invaded  America  and 
became  prevalent  in  China,  Japan,  Aus- 
tralia and  Africa.  Hundreds  of  thou- 
sands of  people  were  infected  and  the 
mortality  was  high.  Since  then  more 
or  less  of  it  has  been  present,  especi- 
ally during  the  winter  months.  Many 
acute^  inflammations  of  the  respira- 
tory mucous  membrane  due  to  pneu- 
mococci  and  streptococci  give  symp- 
toms similar  to  those  produced  by  the 
bacillus  of  influenza,  which  was  final- 
ly isolated  by  Pfeiffer  In  1892  from 
the  purulent  bronchial  secretions  of 
a  patient  suffering  from  the  disease 
and  grown  upon  blood  agar.  The 
Bacillus  of  Influenza  is  an  extremely 
small,  non  motile  organism  of  irreg- 
ular length,  with  rounded  ends,  rarely 
forming  chains.  The  organisms  do 
not  take  the  ordinary  aniline  dyes 
well  and  are  best  demonstrated  by 
staining  with  a  10%  aqueous  fuchsin 
for  5  to  10  minutes,  or  with  Loeffler's 
alkaline  methylene  blue  for  5  minutes. 
They  are  Gram  negative.  Occasional- 
ly a  slight  polar  stain  may  be  ob- 
served. In  the  smears  from  the 
bronchial  secretions,  the  bacilli  lie 
close  together  in  thick,  irregular 
clusters  without  definite  parallelism. 
The  fact  that  they  very  rarely 
form  chains  is  considered  character- 
istic. The  organisms  are  found 
in  the  nasal  passages  and  bronchial 
secretions  of  those  sick  from  the  dis- 
ease. The  organs  affected  most  fre- 
quently in  man  are  the  upper  respira- 
tory passages  and  lungs.  The  disease 
takes  the  form  of  a  broncho  or  lobular 
pneumonia.  The  broncho  pneumonias 
produced  by  the  organism  do  not 
differ  essentially  from  those  produced 
from  other  microorganisms,  conse- 
quently, a  bacteriological  diagnosis  is 
imperative.  The  infection  is  not  in- 
frequently followed  by  abscess  or 
gangrene  of  the  lung,  and  occasional- 
ly a  chronic  interstitial  process  is  de- 
veloped. The  organisms  have  been 
found  in  the  middle  ear,  the  meninges, 
the  brain  and  spinal  cord.  Although 
the  general  character  of  the  symp- 
toms suggests  septicemia,  the  organ- 
ism has  not  been  found  in  the  circu- 
lating blood.  The  incubation  period 
is  short,  having  been  shown  to  develop 


BACTERIOLOGY.  221 

In  24  hours.  The  organism  may  re- 
main in  the  bronchial  secretions  of 
convalescents  or  even  in  the  secre-, 
tions  of  normal  individuals  for  many 
years.  Animals  are  not  susceptible 
to  the  infection,  excepting  the  monkey, 
in  which  influenza-like  symptoms 
have  been  produced  by  rubbing  pure 
cultures  upon  the  unbroken  nasal 
mucosa.  Rabbits  inoculated  intravt^n- 
ously  suffer  from  severe  symptoms 
which  are  probably  purely  toxic.  The 
Immunity  produced  by  an  attack  of 
influenza,  if  any,  is  of  very  short 
duration. 

The  organism  is  aerobic.  The  isolation 
is  not  easy.  PfeifCer  succeeded  in 
growing  the  bacillus  upon  serum  agar 
plates  which  had  been  smeared  with 
the  pus  from  the  bronchial  secretions 
of  patients.  Agar  smeared  with  blood 
is  a  favorable  media  for  its  growth. 
The  organism  grows  well  symbiotic- 
ally  with  staphylococci  which  seem 
to  create  a  favorable  environment  for 
their  development.  The  organism 
does  not  grow  at  room  temperature 
at  Z1V2°  C,  and  on  favorable  media, 
the  colonies  appear  in  from  18  to  24 
hours  as  minute,  colorless,  transpar- 
ent, discrete  droplets.  In  order  to 
keep  the  cultures  alive  they  should 
be  stored  at  room  temperature  and 
transplanted  every  four  or  five  days. 
The  organism  is  very  sensitive  to 
heat,  desiccation  and  disinfectants. 

^CNFIiUENZA-I^IKE  BACIUI. 

Fseudo  Influenza  Bacillus.  This  organ- 
ism was  found  by  Pfeiffer  in  the 
broncho  pneumonic  process  of  chil- 
dren. It  is  slightly  larger  than  the 
bacillus  of  influenza,  non  motile  and 
Gram  negative,  with  tendency  to  form 
threads  and  involution  forms.  It  is 
strictly  aerobic.  Woolstein  believes 
the  organism  to  be  the  same  as  the 
bacillus  of  influenza,  after  having 
studied  it  both  culturally  and  by  ag- 
glutination tests.  Strains  of  similar 
bacilli  have  been  isolated  from  cases 
of  pertussis. 

Koch-Weeks  Bacillus.  This  bacillus  was 
described  by  Koch  in  1883  and  Weeks 
in  1887  in  connection  with  an  acute 
conjunctival    inflammation.  Morph- 


222  BACTERIOLOGY. 


ologically  it  resembles  the  bacillus  of 
influenza,  though  more  slender  and  of 
greater  length.  It  grows  at  a  tem- 
perature of  371/^°  C.  only,  and  can  be 
cultivated  upon  serum  or  ascitic  fluid 
without  hemoglobin,  in  which  respect 
it  differs  from  the  bacillus  of  in- 
fluenza. It  is  Gram  negative. 
The  Bacillus  of  Fleuro  Fneimioiila  of 
Babbits  is  a  small  Gram  negative  ba- 
cillus slightly  larger  than  the  bacillus 
of  influenza  and  grows  upon  ordinary 
media.  The  organism  was  described 
by  Beck. 

The  Bacillus  Murisepticus  and  Bacillus 
Bhusiopathiae  are  organisms  morph- 
ologically similar  to  the  influenza 
group  but  can  be  easily  separated 
from  them  because  of  their  luxuriant 
growth  on  ordinary  media.  They  are 
more  closely  related  to  the  hemor- 
rhagic septicemic  group  of  organisms. 

BACZIil^US  BOBDET-GBNGOir. 

(Bacillus  of  Whoopiugr  Coug'h). 

This  organism  was  discovered  by  Bordet 
and  Gengou  in  the  sputum  of  a  child 
ill  with  pertussis.  It  is  as  yet  not 
positive  as  to  the  specificty  of  this 
organism  for  whooping  cough.  Cul- 
'tivation  was  not  successfully  carried 
out  until  1906.  Since  then  almost 
pure  cultures  of  the  organism  have 
been  obtained  during  the  early  par- 
oxysm of  the  disease,  and  for  this 
reason  it  is  thought  likely  to  be  a 
speciflc  cause.  In  early  cases,  true 
influenza  bacilli  have  often  been 
found,  and  these  seem  to  remain  in 
the  sputum  of  such  patients  for  a 
longer  period  and  in  larger  numbers 
than  the  bacillus  of  Bordet-Gengou. 
The  organism  though  slightly  larger 
than  influenza  bacillus  resembles  it 
greatly,  but  shows  some  morpholog- 
ical differences  and  less  tendensy  to 
pleomorphism.  It  is  a  small  ovoid 
bacillus  found  scattered  in  enormous 
numbers  among  the  pus  cells,  some- 
times within  the  cell,  in  the  sputum 
early  in  the  disease.  Occasionally  it 
resembles  a  micrococcus,  though  gen- 
erally the  form  is  constant,  slightly 
enlarged  individuals  may  be  encount- 
ered. The  poles  of  the  cell  may  stain 
more  deeply  than  the  center.  It.  is 
stained  with  alkaline  methylene  blue, 
dilute  carbofuchsin  or  aqueous  fuchsin. 


BACTERIOLOGY. 


223 


It  is  decolorized  by  Gram.  The  group- 
ing is  separated,  though  sometimes  in 
end  to  end  pairs. 

Inoculated  into  the  respiratory  tract  of 
monkeys  it  has  failed  to  produce  the 
disease.  One  to  two  cc.  of  an  extract 
made  by  emulsifying  the  agar  growths 
with  a  little  salt  solution,  dried  in 
vacuo  and  ground  in  the  mortar,  di- 
luted with  salt  solution  and  centri- 
fugalized  and  decanted,  and  injected 
intravenously  into  rabbits,  will  kill 
within  24  hours.  A  subcutaneous  in- 
oculation will  produce  necrosis  and 
ulceration  without  marked  constitu- 
tional symptoms.  Specific  agglutinins 
obtained  from  immunized  animals 
distinguishes  the  organism  from  that 
of  the  bacillus  of  influenza. 

It  is  cultivated  from  the  sputum  on  a 
medium  made  by  adding  100  gms.  ol 
sliced  potato  to  200  cc.  of  4%  watery 
solution  of  glycerine,  steamed  in  an 
autoclave;  50  cc.  of  this  extract  is 
mixed  with  150  cc.  of  6%  salt  solu- 
tion, then  5  gms.  of  agar  are  added. 
It  is  now  melted  in  an  autoclave  and 
filled  in  quantities  of  2  to  3  cc.  into 
test  tubes  and  sterilized.  To  each 
tube  is  now  added  an  equal  volume 
of  sterile  defibrinated  rabbit's  or  hu- 
man blood.  The  substance  is  mixed 
and  the  tube  slanted.  On  such  a 
media,  growth  appears  after  24  to  48 
hours  as  small  greyish  rather  thick 
colonies.  The  second  generation  on 
this  media  becomes  rapid  and  lux- 
uriant and  after  several  generations, 
they  resemble  the  growth  of  the 
typhoid  bacillus.  Later,  it  seems  less 
dependent  upon  the  presence  of 
hemoglobin  than  does  the  bacillus  of 
influenza.  It  will  develop  at  as  low 
a  temperature  as  5°  C.  but  grows 
best  at  371/2°  C. 

BACIl^IiUS  MORAX-AXENFEI^D. 

The  Bacillus  of  Morax-Axenfeld  was 
discovered  by  Morax  in  1896  from  a 
type  of  chronic  catarrhal  conjuncti- 
vitis, which  attacked  both  eyes,  espe- 
cially in  the  angles  of  the  eye  and 
most  severe  at  or  about  the  caruncle. 
The  swelling  produced  is  not  great 
and  there  is  hardly  ever  ulceration. 
The  condition  becomes  subacute  and 


224  BACTERIOLOGY. 

chronic  and  may  be  diagnosed  by- 
smear  preparations  made  trom  the 
pus,  which  is  especially  abundant 
during  the  night.  The  organism  is  a 
short  thick  diplobacillus.  It  may, 
however,  appear  singly  or  in  short 
chains.  The  ends  are  rounded,  the 
center  slightly  bulged.  They  are 
stained  by  the  aniline  dyes  and  de- 
colorized by  grams.  Inoculation  with 
pure  cultures  has  produced  subacute 
conjunctivitis  in  man.  The  produc- 
tion of  lesions  in  lower  animals  has 
been  unsuccessful. 
The  organism  is  cultivated  only  upon 
alkaline  media  containing  blood  or 
blood  serum.  At  the  end  of  24  hours 
on  LoefHer's  blood  serum,  small  areas 
of  liquefaction  are  noticed,  later  the 
entire  media  is  liquefied.  Upon  serum 
agar,  delicate,  greyish,  droplike  col- 
onies, not  unlike  those  of  the  gono- 
coccus,  are  formed. 

BACIUUS  OF  ZUB  NEEDEN. 

Zur  Needen  described  a  small,  slightly 
curved,  non  motile  diplobacillus  in 
ulcerative  conditions  of  the  cornea, 
to  which  he  attributed  etiological  im- 
» portance.  The  organism  is  stained 
by  the  ordinary  aniline  dyes,  though 
poorly  at  the  ends  and  decolorized  by 
Gram's. 

Corneal  ulcers  are  produced  by  inocula- 
tion of  guinea  pigs. 

Upon  agar  within  24  hours,  trans- 
parent, slightly  fluorescent,  round, 
raised,  rather  coarsely  granular  col- 
onies are  formed  which  show  a  tend- 
ency to  confluence.  Gelatin  is  not 
liquefied.  Milk  is  coagulated.  Upon 
potato,  there  appears  a  thick,  yellow- 
ish growth.  Upon  dextrose  media, 
there  is  acid  formation  but  no  gas. 
Indol  is  not  produced  in  peptone. 
Bacillus  of  DxLcrey. 
(Bacillus  of  Soft  Chancre). 

This  bacillus  was  first  described  and 
obtained  in  pure  culture  by  Ducrey  in 
1889.  It  produces  a  lesion,  which  oc- 
curs usually  upon  the  genitals  or 
the  skin  surrounding  the  genitals 
of  an  acute.  Inflammatory,  destructive 
nature  and  called  "soft  chancre"  or 
"chancroid."  The  lesion  begins  usual- 
ly, as  a  small  pustule,  which  soon 
ruptures  and  forms  a  small,  round. 


BACTERIOLOGY.  225 


depressed,  irregular  ulcer,  with  under- 
mined edges  and  a  necrotic  floor,  dis- 
charging seropurulent  fluid  which  is 
extremely  infectious.  This  ulcer 
spreads  rapidly  and  leads  usually  to 
lymphatic  swellings  in  the  groin, 
which  later  give  rise  to  abscesses 
spoken  of  as  "buboes."  The  lesion 
differs  from  the  syphiltic  chancre  in 
that  there  is  no  induration. 
It  is  an  extremely  small,  non-motile 
bacillus,  generally  appearing  in  short 
chains  and  in  parallel  rows,  though 
it  may  be  found  irregularly  grouped. 
It  stains  easily  though  irregularly 
with  aniline  dyes.  More  stained  at 
the  poles,  decolorized  by  Gram's.  The 
bacilli  are  found  in  the  pus,  often 
within  the  leucocyte.  Various  in- 
vestigators have  succeeded  in  pro- 
ducing lesions  in  man  by  inoculating 
with  pure  cultures.  Attempts  to  in- 
oculate animals  have  beeja  unsuccess- 
ful. 

The  organism  grows  on  agar  medium  to 
which  blood  has  been  added.  Coag- 
ulated blood  kept  for  several  days 
in  sterile  tubes  is  a  very  favorable 
medium.  The  organism  is  isolated 
by  aspirating  an  unruptured  bubo 
with  a  sterile  hypordermic  syringe 
and  transferring  the  pus  in  quantities 
directly  to  the  agar.  If  no  buboes 
are  present,  the  primary  lesion  may 
be  cleansed  with  water  or  salt  solu- 
tion, material  scraped  from  the  bot- 
tom of  the  ulcer  by  means  of  a  stiff 
platinum  loop  and  smeared  over  the 
the  surface  of  blood  agar  plates. 
After  a  period  of  48  hours,  small, 
transparent,  grey,  finely  granular, 
isolated  colonies  appear  upon  the 
agar  plate.  These  rarely  grow  larger 
than  pin  head  size.  The  cultures  die 
readily  at  room  temperature  but  may 
be  kept  alive  in  the  incubator  for  a 
week  or  more. 

SFIBIIiI^UM  CHOLERAS  ASIATICAE. 
(The  Comma  Bacillus  of  Koch). 

This  organism  was  discovered  by  Kocn 
in  1883  in  the  dejecta  of  patients  suf- 
fering from  Asiatic  cholera. 

Asiatic    Cholera,    a    disease    occurring  s 
spontaneously    only    in    man,    is  en- 
demic   in    eastern    countries,  partic- 
ularly India.     From  time  to  time  it 


226  BACTERIOLOGY. 


has  become  epidemic  in  Europe  and 
Asia,  not  infrequently  sweeping  over 
almost  the  entire  earth.  The  last 
great  epidemic  began  about  1883,  dur- 
ing which  time  there  were  800,000 
victims  in  Russia  alone,  and  reached 
Germany  in  1892.  Prom  there  it  en- 
tered America  and  Africa. 
The  vibrio  or  spirillum  of  cholera  is  a 
small,  curved  rod.  The  curvature  may 
vary  from  the  comma  like  form  to  a 
distinct  corkscrew  like  spiral,  with 
one  or  two  turns.  It  is  actively 
motile  by  reason  of  a  single  polar 
flagellum.  The  comma  form  predom- 
inates in  young  cultures,  while  the 
longer  forms  are  more  numerous  in 
old  cultures.  Prolonged  artificial  cul- 
tivations without  passage  through 
the  animal  body  tend  to  change  the 
form  of  the  organism  into  a  straight 
type.  They  take  the  ordinary  watery 
aniline  dy^s  well  and  are  decolorized 
by  gram.  The  infection  is  essentially 
of  the  intestine  and  contracted  by 
the  injections  of  the  organisms  with 
water,  food  or  contaminated  material. 
A  few  organisms  entering  into  the 
stomach  may  be  checked  by  the  nor- 
mal gastric  secretions  by  reason  of 
the  organism's  sensitiveness  to  an 
acid  reaction.  Entering  the  intestine, 
however,  they  proliferate  and  rapidly 
outgrow  the  normal  intestinal  flora. 
Autopsies  show  extreme  congestion 
of  the  intestinal  walls,  with  occa- 
sional ecchymosis  and  localized  ne- 
crosis of  the  mucosa,  with  swelling 
of  the  solitary  lymph-follicles  and 
Peyer's  patches.  The  organisms 
penetrate  the  mucosa  and  lie  within 
it  in  the  layers  next  to  the  submucosa. 
The  intestines  are  filled  with  watery, 
slightly  bloody,  or  "rice  water"  stools, 
which  is  characteristic  and  from 
which  pure  cultures  of  the  organism 
may  be  isolated.  They  are,  in  fact, 
found  only  in  the  intestines  and  their 
contents.  The  parenchymatous  de- 
generation in  other  organs  is  of  toxic 
origin.  In  animals  the  disease  never 
appears  spontaneously  so  that  special 
methods  were  necessary  in  order  to 
produce  the  disease  experimentally. 
Subcutaneous  inoculations,  unless  in 
large  quantities  of  the  organism,  in 
rabbits  and  guinea  pigs  very  seldom 


BACTERIOLOGY.  227 


produce  more  than  a  slight  illness. 
Intraperitoneal  inoculation,  if  in 
proper  quantities,  generally  leads  to 
death.  Different  strains  of  the  cholera 
spirillum  vary  greatly  in  their  viru- 
lence and  this  may  be  enchanced  by 
repeated  passage  through  animals. 

The  poisonous  action  of  the  cholera  or- 
ganism depends  upon  the  formation 
of  true  secretory  toxins  and  upon 
endotoxins.  Which  of  these  is  para- 
mount in  producing  the  disease  can- 
not be  stated  definitely. 

Active  immuniation  is  accomplished  by 
inoculation  of  dead  cultures  or  small 
doses  of  living  bacteria.  One  attack 
confers  protection  against  subsequent 
infections.  Specific  bacteriolysins  and 
agglutinins  are  found  in  the  serum  of 
immunized  animals,  which  are  of 
great  importance  in  making  a  bac- 
teriological diagnosis  of  the  true  or- 
ganism. For  protective  inoculation 
of  man,  see  ''Cholera  Vaccine." 

The  organism  is  aerobic  and  facultative 
anaerobic.  It  grows  between  22°  C. 
and  40°  C,  with  an  optimum  of  ZTY2° 
C.  The  method  of  isolation  is  ac- 
complished by  inoculating  a  set  of 
gelatin  plates,  a  set  of  agar  plates 
and  a  set  of  Dunham's  peptone  tubes 
with  the  suspected  material.  If  pres- 
ent in  great  quantities,  they  may  be 
picked  up  from  the  plate  colonies. 
When  less  numerous,  they  may  be 
found  in  the  topmost  layers  of  the 
peptone  broth  after  8  or  10  days  at 
371/^°  C,  from  which  plate  dilutions 
can  be  prepared  and  the  colonies 
picked  up  and  identified  by  means  of 
cultural  and  agglutinative  tests. 

The  organism  grows  on  all  the  ordinary 
culture  media  of  moderate  alkalinity. 
Slight  acidity  will  not,  however,  pre- 
vent growth.  In  gelatin  plates  at 
room  temperature,  yellowish  grey  pin 
head  colonies  appear  within  24  hours. 
The  colonies  increase  in  size;  the 
gelatin  becomes  liquefied.  Magnified 
the  colonies  appear  coarsely  granular 
with  irregular  margins.  In  the 
gelatin  stab,  the  liquefaction  is  fun- 
nel shaped.  Upon  agar  plates,  grey- 
ish opalescent  colonies  appear  within 
18  to  24  hours.  These  are  easily 
""differentiated,  from  other  bacteria  of 
the  feces,  by  reason  of  their  trans- 


228  BACTERIOLOGY. 


parency.  Coagulated  blood  serum  is 
liquefied.  On  potato,  the  growth  is 
heavy  and  of  a  brownish  color.  Milk, 
the  growth  is  heavy  without  coagu- 
lation. In  broth,  there  is  a  general 
clouding  with  pellicle  formations.  In 
Dunham's  peptone,  indol  is  produced. 
The  organism  is  not  very  resistant  to 
drying.  Boiling  destroys  them  im- 
mediately. They  are  killed  after  an 
hour's  exposure  to  a  temperature  of 
60°  C.  The  common  disinfectants,  in 
very  weak  solution,  will  destroy  them 
after  a  short  exposure.  The  organism 
frozen  in  ice  may  live  for  three  or 
four  days. 

ORGANISMS   AZiIiIED    TO  CHOZkERA 
SFIBH^IiVM. 

The  examination  of  the  stools  of  per- 
sons suffering  from  cholera  have  re- 
vealed, in  a  small  percentage  of  cases, 
spirilla  that  somewhat  closely  re- 
semble the  true  cholera  organisms, 
and  they  are  of  bacteriological  im- 
portance by  reason  of  the  difficulty 
which  they  add  to  the  work  of  dif- 

.  ferentiation.  Some  bear  only  morph- 
ological resemblance,  while  others 
can  be  distinguished  from  the  true 
cholera  organism  only  by  the  serum 
reactions  and  the  pathogenicity  to 
animals. 

The  Spirillum  Metchnikovl  was  dis- 
covered by  Gamaleia  in  1888,  in  the 
intestinal  contents  and  the  blood  of 
fowls  dying  of  an  infectious  disease, 
which  prevailed  in  certain  parts  of 
Russia  during  the  summer  months, 
and  which  presents  symptoms  resem- 
bling fowl  cholera.  It  is  identical 
with  the  spirillum  of  cholera  in  its 
morphological  and  staining  reactions. 
It  possesses  a  single  polar  flagella 
and  is  actively  motile.  Culturally,  it 
is  similar  to  the  cholera  spirillum,  ex- 
cept for  more  luxuriant  growth  and 
more  rapid  liquefaction  of  gelatin.  It 
also  gives  the  indol  reaction  in  pep- 
tone media.  Differentiation  from  the 
cholera  spirillum  is  made  by  inocu- 
lating minute  quantities  subcutane- 
ously  into  pigeons,  producing  there- 
by a  rapidly  fatal  septicemia.  It  is 
more  pathogenic  for  guinea  pigs  than 
is  the  cholera  spirillum.    There  is  no 


BACTERIOLOGY.  229 


lysis  or  agglutination  by  cholera  im- 
mune serum. 

The  Spirillum  of  Pinkler-Prior  was  Iso- 
lated by  Pinkler  and  Prior  in  1884 
from  the  feces  of  patients  having 
cholera  nostras.  It  is  like  the  true 
cholera  spirillum,  though  somewhat 
longer  and  thicker  and  less  uniformly 
curved  and  not  so  uniform  in  dia- 
meter, the  central  portion  being 
usually  wider  than  the  pointed  ends. 
Culturally,  it  resembles  the  cholera 
spirillum  except  that  its  growth  is 
more  rapid  and  thick  upon  the  or- 
dinary culture  media.  It  does  not 
form  indol  in  peptone  solutions  nor 
does  it  give  specific  serum  reaction 
with  cholera  immune  serum. 

Tlie  Spirillum  Massauali  was  isolated 
by  Pasquale  in  1891  at  Massauah 
from  a  doubtful  case  of  cholera.  In 
its  pathogenicity,  it  closely  resem- 
bles the  spirillum  Metchnikovi  in  that 
it  is  able  to  produce  septicemia  in 
pigeons.  Culturally  and  morpholog- 
ically it  resembles  the  cholera  spirul- 
lum.  It  possesses  four  flagella  and 
does  not  give  specific  serum  reaction 
with  cholera  immune  serum. 

The  Spirillum  Deneke  was  isolated  from 
butter  by  Deneke.  It  greatly  resem- 
bles the  spirillum  of  Finkler-Prior. 
It  does  not  produce  indol  in  peptone 
media. 

ACID  FAST  GROUP  OF  ORGANISMS. 

This  so-called  "acid  fast"  group  of  or- 
ganisms derives  their  name  by  the 
non-permeability  of  the  ordinary 
stains  unless  exposed  to  them  for  a 
long  time  or  by  heating  the  solutions. 
The  stain  having  entered  the  organ- 
ism will  retain  it  even  when  treated 
with  alcohol  and  strong  acids.  The 
acid  fast  nature  seems  to  depend 
upon  the  fatty  substances  contained 
within  the  organisms. 

Tlie  Bacillus  of  Tuberculosis. 

This  bacillus  was  isolated  by  Koch  in 
1882  and  established  the  etiological 
relationship  of  the  bacillus  to  the 
disease  by  infecting  guinea  pigs  and 
other  animals  with  pure  cultures  of 
the  bacillus,  and  producing  the  charac- 
teristic lesions.  Previous  to  this  tne 
transmission  of  tuberculous  material 
was  accomplished  by  Klenkce  in  1843 


230  BACTERIOLOGY. 


and  Willeman  in  1865  and  the  tubercle 
bacillus  had  been  demonstrated  in 
tissue  sections  by  Baumgarten  early 
in  1882. 

The  tubercle  bacillus  is  a  slender, 
straight,  or  slightly  curved  rod  usual- 
ly rounded  at  the  ends.  The  diameter 
may  be  uniform  throughout,  thougn 
more  often  they  appear  beaded  and 
irregularly  stained.  This  irregular- 
ity in  staining  generally  appears  in 
old  cultures  and  may  be  regarded  as 
vacuola.  The  bodies  of  the  bacilli 
may  bulge  slightly  in  three  or  four 
places,  presenting  oval  or  round 
knobs  which  take  the  stain  deeply 
and  are  very  resistant  to  decolorization. 
A  cell  membrane  has  been  described 
which  confers  resistance  to  the  or- 
ganism against  drying,  etc.  It  gives 
a  cellulose  reaction  and  the  waxy 
material  obtained  from  the  culture  by 
extraction  is  believed  to  be  contained 
within  it.  Branched  forms  of  the  or- 
ganism have  been  demonstrated  by 
various  observers,  and  by  reason  of 
this  fact,  it  is  probable  that  the 
tubercle  bacillus  is  not  a  member  of 
the  family  schizomycetes  but  belongs 
to  the  higher  bacteria. 

The  bacilli  do  not  stain  easily  with  the 
ordinary  aniline  dyes.  They  must  be 
exposed  to  the  stains  for  a  long  time 
or  the  stain  solution  must  be  heated. 
After  having  been  stained,  however, 
they  are  extremely  resistant  to  acids 
and  decolorizations.  For  methods  of 
staining  see  section  on  Staining 
Formulas. 

Very  young  cultures  are  often  not  acid 
fast  and  it  is  not  always  possible  to 
demonstrate  acid  fast  bacilli  in  pus 
from  cold  abscesses,  in  sputum,  in 
serous  exudates  and  in  lesions  of  the 
lymph  nodes  which  can  be  shown  by 
animal  inoculation  to  be  tuberculant. 
In  this  material.  Much  calls  attention 
to  Gram  positive  granules  arranged 
singly  in  short  chains  or  irregular 
clumps,  which  he  believed  to  be  non 
acid  fast  tubercle  bacilli.  There  is  no 
doubt  as  to  the  truth  of  his  belief  in 
that  his  work  has  repeatedly  been 
confirmed.  These  Gram  positive 
bodies,  however,  are  not  of  great  di- 
agnostic value  as  other  bacilli  form 
granules    of    the    same  appearance. 


BACTERIOLOGY.  231 


Small  rods  and  splinters  are  also 
found  which  are  Gram  positive  and  do 
not  stain  by  the  carbofuchsin  method. 
Other  organisms  of  the  acid  fast 
group,  which  may  be  difficult  to  dif- 
ferentiate from  the  tubercle  bacillus, 
are  the  bacillus  of  leprosy  and  the 
smegma  bacillus.  By  reason  of  the 
distribution  of  the  smegma  bacillus 
in  feces,  urine  or  even  sputum,  it  be- 
comes necessary  to  apply  to  sus- 
pected specimens  the  other  stains 
which  are  devised  for  the  differenti- 
ation of  this  bacillus  from  that  of 
tuberculosis.  Pappenheim's  stain  is 
the  one  most  frequently  employed  for 
thiis  purpose.  Stained  by  Pappen- 
heim's method  the  tubercle  bacilli  re- 
main red;  the  smegma  bacilli  appears 
blue.  Tubercle  bacilli  are  Gram  posi- 
tive. When  tubercle  bacilli  are  pres- 
ent in  extremely  small  numbers  in 
the  sputum  and  other  material,  it 
may  be  impossible  to  find  them  by 
direct  examination,  and  often  the 
only  method  of  demonstrating  them 
will  be  by  animal  inoculation.  Meth- 
ods of  concentration  have  been  de- 
vised by  which  the  bacilli  may  be 
found  when  only  a  few  are  present. 
One  method  is  to  add  hydrogen  per- 
oxide to  the  sputum.  This  dissolves 
the  mucous  and  allows  the  solid 
particles  to  settle  by  centrifugation. 
Another  method,  much  used  today,  is 
by  the  use  of  "antiformin,"  which  ia 
made  up  of  equal  parts  of  liquor 
soda    chlorinated    (sodium  carbonate 

^  600  parts,  chlorinated  lime  400  parts 
and  distilled  water  4,000  parts)  and 
a  15%  solution  of  caustic  soda.  The 
sputum  is  pored  into  a  10  to  15%  so- 
lution of  antiformin  and  allowed  to 
stand  for  several  hours.  The  other 
elements  of  the  sputum,  as  the  cells 
and  bacteria,  will  be  dissolved  out, 
leaving  only  the  acid  fast  bacteria  in 
the  residue.  The  tubercle  bacilli  are 
not  killed  by  this  process  and  after 
sufficient  washing,  they  may  be  cul- 
tivated or  can  produce  lesions  in 
guinea  pigs. 

The  organism  produces  in  man  and 
susceptible  animals,  a  specific  phe- 
nomenon of  inflammatory  foci,  known 
as  tubercles.  Tuberculosis  is  the 
most  common  disease  in  man.  ISTagel 


232  BACTERIOLOGY. 


in  a  large  series  of  autopsies  found 
lesions  of  healed  or  active  tubercu- 
losis in  a  large  percentage  of  cases. 
The  disease  is  less  common  in  the 
rural  districts  than  in  large  towns. 
The  pulmonary  infection  is  the  most 
common  type  in  man,  though  tuber- 
culosous  process  may  be  found  in  the 
skin,  the  bones,  the  joints,  the  or- 
gans of  special  sense,  the  abdominal 
viscera,  and  peritoneum.  Infection 
takes  place  by  inhalation  or  through 
the  skin,  or  through  the  digestive  ap- 
paratus. Behring  has  caused  a  great 
deal  of  discussion  by  stating  that  he 
believed  a  large  percentage  of  all 
cases  of  tuberculosis  originated  in 
childhood  by  way  of  the  intestinal 
tract.  He  therefore  brought  to  notice 
the  problem  of  the  virulence  of  bovine 
tubercle  bacilli  for  human  beings,  as 
he  assumed  that  the  infection  of 
children  is  due  to  the  use  of  infected 
milk.  From  the  contributions  of 
Parke  and  Krumweide,  it  would  seem 
that  human  adults  are  relatively  In- 
susceptible to  bovine  infection  which 
may  take  place,  but  is  unusual. 

A  more  relative  susceptibility  is  found 
under  16  years  of  age,  and  the  danger 
of  milk  infection  is  without  doubt 
great;  in  fact,  one  third  of  the  cases 
arising  from  this  source.  The  danger 
of  bovine  tuberculosis  is  greatest 
under  5  years  of  age. 

The  above  statement  would  indicate 
that  Behring's  original  statement 
cannot  be  upheld,  though  it  does, 
without  doubt,  point  to  the  great 
dangers  of  milk  infections. 

The  tubercle  bacillus  (human)  is  patho- 
genic for  guinea  pigs;  less  so  for 
rabbits  and  still  less  so  for  dogs.  It 
is  slightly  pathogenic  for  cattle. 

The  work  connected  with  the  isolation 
of  specific  toxins  has  led  to  a  chem- 
ical analysis  of  the  organism,  which 
shows  it  to  consist  of  about  8Sy2% 
of  water;  20  to  26%  of  the  residue 
can  be  extracted  with  ether  and  al- 
cohol. This  material  consists  of  fat- 
ty acids  and  waxy  substance  (fatty 
acids  in  combination  with  higher  al- 
cohol). The  residue,  after  the  al- 
cohol ether  extraction,  is  made  up 
chiefly  of  proteids,  which  can  be  ex- 
tracted with  dilute  alkaline  solutions 


BACTERIOLOGY.  233 


and  consist  principally  of  nucleo  al- 
bumens, a  fraction  of  which  Is  sus- 
pected of  being  the  pathogenic  prin- 
cipal of  the  bacillus  in  that  it  shows 
high  toxicity.  The  remainder  con- 
tains cellulose,  representing  probably 
the  frame  work  of  the  cell  membrane 
and  the  ash  which  is  rich  in  calcium 
and  magnesia. 

(For  the  toxins  and  their  method  of 
preparation  see  Tuberculins).  Num- 
erous attempts  have  been  made  to 
passively  immunize  tubercular  sub- 
jects with  the  sera  of  immune  an- 
imals. The  methods  most  used  for 
the  production  of  such  serum  is  that 
of  Maragliano,  who  believes  that 
there  is  a  toxic  albumin  in  the  cul- 
tures of  the  tubercle  bacilli,  which  is 
destroyed  by  the  heating  employed  in 
the  usual  production  of  tuberculins. 
He  therefore  prepares  his  substance 
by  filtering  unheated  cultures  and 
precititates  the  filtrates  with  alcohol. 
He  now  makes  a  watery  extract  of 
the  bacillary  bodies  and  with  these 
two  substances  he  immunizes  horses. 
After  4  to  6  months  of  treatment,  he 
withdraws  blood  from  the  horse  and 
separates  the  serum.  This  serum 
called  Maragliano's  serum  is  exten- 
sively used  in  Italy.  Its  value  is 
very  doubtful. 

Marmorex  claims  that  the  toxin  pro- 
duced by  the  bacillus  of  tuberculosis 
is  dependent  to  a  great  extent  upon 
the  medium  on  which  It  Is  grown. 
He  believed  that  the  substance  ob- 
tained in  tuberculin  was  not  true 
toxins  of  the  bacillus  and  that  the 
true  toxins  could  only  be  elaborated 
by  a  younger  phase  of  the  bacillus, 
as  it  occurs  within  the  animal  body 
or  on  media  composed  of  animal  tis- 
sue. He  therefore  grows  his  cultures 
on  a  medium  of  leucotoxic  serum  and 
liver  tissue.  Such  cultures  he  be- 
lieves to  contain  no  tuberculant.  The 
sera  produced  by  immunization  with 
these  cultures  is  supposed  by  him  to 
have  high  curative  powers. 

The  organism  is  aerobic  with  a  temper- 
ature range  of  30°  to  42°  C.  with  an 
optimum  of  37%.  The  organism  is 
not  easily  cultivated.  Isolation  from 
tuberculous  material  is  materially 
aided  by  inoculation  into  guinea  pigs. 


234  BACTERIOLOGY. 


The  animals  often  withstand  the  acute 
infection  produced  by  contaminating 
organisms  and  in  4  to  6  weeks  die 
from  the  tuberculous  infection.  The 
bacilli  may  then  be  cultivated  from 
the  lymphnodes  or  other  foci  which 
contain  only  tubercle  bacilli.  Koch 
isolated  the  organism  from  the 
sputum  as  follows: — 
The  sputum  is  thoroughly  washed  in 
running  water  to  free  it  from  mucus. 
It  is  then  washed  in  8  or  10  changes 
of  sterile  water.  The  material  for 
cultivation  is  taken  from  the  center 
of  the  washed  mass,  if  possible. 
Select  the  caseous  material,  which  is 
often  present  in  such  sputum,  and 
either  inoculate  culture  media  direct- 
ly or  inoculate  animals  as  indicated 
above.  On  blood  serum,  at  the  end 
of  8  to  14  days,  small,  dry,  greyish 
white,  scaly  colonies  with  corrugated 
surfaces  appear.  At  the  end  of  3  to 
4  weeks'  cultivation  these  colonies 
joined  together  cover  the  surface  of 
the  medium  as  a  dry,  whitish,  wrin- 
kled membrane.  The  organism  will 
grow  well  on  agar  slants  to  which 

1  to  2  cc.  of  rabbit's  blood  has  been 
•added.    Glycerine  agar  is  a  favorable 

media  for  its  growth.  Glycerine 
bouillon  of  a  slightly  alkaline  reac- 
tion is  an  excellent  culture  medium. 
This  medium  is  placed  in  thin  layers 
into  wide  mouthed  flasks  and  then  in- 
oculated by  carefully  floating  flakes 
of  the  culture  upon  the  surface.  The 
organism  will  appear  as  a  thin, 
opaque,  floating  film,  which  rapidly 
thickens  into  a  white,  wrinkled,  or 
granular  layer,  spreading  out  in  all 
directions  and  covering  the  entire  sur- 
face of  the  bouillon  in  from  4  to  6 
weeks.  Later,  portions  of  the  mem- 
brane will  sink  to  the  bottom.  The 
organism  will  also  grow  freely  upon 
glycerine  potato. 
The  life  of  cultures  kept  in  a  favorable 
environment  (access  of  oxygen)  is 
from  2  to  8  months.  In  sputum,  they 
remain  alive  and  virulent  for  6  weeks, 
and  in  dried  sputum  for  more  than 

2  months. 

In  fluid  media,  the  organism  is  killed 
at  a  temperature  of  60°  C.  in  from 
15  to  20  minutes,  and  at  80°  C.  in  ^ 
minutes;  at  90"   C.   in    from   1  to 


BACTERIOLOGY.  235 

minutes.  They  withstand  dry  heat  at 
a  100°  C.  for  1  hour.  They  are  re- 
sistant to  cold;  5%  carbolic  acid  will 
kill  the  organism  in  a  few  minutes. 
If  used  as  a  disinfectant  for  sputum 
by  reason  of  the  fact  that  the  mucous 
coat  will  protect  the  bacilli,  the  dis- 
infectant should  be  allowed  to  act  for 
5  to  6  hours.  Direct  sunlight  will 
kill  the  organism  in  a  few  hours. 

The  Bacillus  of  Bovine  Tuberculosis. 
The  difference  between  the  reaction  to 
the  infection  in  the  bovine  type  of  the 
disease  and  that  of  man  was  studied 
by  Koch  in  his  early  work.  He 
thought  this  difference  to  be  due  to 
the  nature  of  the  infected  subjects. 
Theobal  Smith,  in  1898,  pointed  out 
the  difference  between  the  bacilli  iso- 
lated from  man  and  those  isolated 
from  cattle.  He  determined  that  the 
bovi^p  bacilli  were  usually  shorter 
than  the  human  bacilli  and  they  did 
not  grow  as  well  upon  artificial  media. 
Grown  upon  slightly  acid  glycerine 
bouillon,  the  bovine  organism  will  re- 
duce the  acidity  until  a  neutral  or 
slightly  alkaline  reaction  is  reached. 
It  differs  in  this  respect  from  the 
human  bacillus,  which  produces  but 
slight  reduction  of  the  acidity  during 
the  first  week  of  growth.  After  this, 
the  acidity  increases  and  never  reaches 
neutrality.  Marked  differences  have 
also  been  shown  to  extgt  in  the  patho- 
genicity of  these  org'anisms  towards 
various  animal  species.  Guinea  pigs 
die  more  quickly  and  show  more  ex- 
tensive lesions  when  inoculated  with 
the  bovine  type  than  when  inocu- 
lated with  the  human  bacillus.  The 
bovine  bacillus  will  kill  a  rabbit  with- 
in 2  to  5  weeks.  The  human  bacillus 
produces  a  mild  disease,  which  lasts 
frequently  for  6  months,  and  at  times 
fails  to  kill  a  rabbit  at  all. 

Many  attempts,  with  little  or  no  suc- 
cess, have  been  made  to  infect  cattle 
with  the  human  organism.  Infection 
of  the  human  individual  with  the 
bacillus  of  the  bovine  type  has  been 
reported  and  proven  by  Smith,  Parke, 
Krumweide  and  others. 

Bacillus  of  Avian.  Tuberculosis.  Koch 
discovered  bacilli  in  the  lesions  of  dis- 
eased fowl,  which  closely  resembled 
the  bacillus  of  tviberculosis.  Nocard 


236  BACTERIOLOGY. 

and  Roux  differentiated  these  bacilli 
as  a  definite  species.  In  its  staining 
characteristics  and  in  its  morphology, 
it  is  almost  identical  with  the  human 
bacillus  of  human  tuberculosis.  Cul- 
turally, it  differs,  however,  in  that 
growth  is  more  rapid  and  takes  place 
at  a  temperature  of  41*'  to  45"  C. 
Guinea  pigs  are  very  resistant  to  this 
type,  while  rabbits  die  quickly  from 
the  avian  tuberculosis.  Prolonged 
cultivation  and  passage  through  the 
mammalian  bodies  will  cause  the  or- 
ganism to  approach  the  mammalian 
type.  Nocard  has,  on  the  other  hand, 
succeeded  in  rendering  the  mam- 
malian tubercule  bacilli  pathogenic 
for  fowl  by  keeping  them  enclosed  in 
celloidln  sacs  within  the  periton- 
eum of  chickens  for  6  months. 
TulberciQosis  of  Cold  Blooded  Animals. 
A  bacillus  resembling  the  bacillus  of 
tuberculosis  in  its  morphology  and 
acid  fastness  was  isolated  by  Dubarre 
and  Terre  from  cold  blooded  animals, 
as  fish,  frogs,  turtles  and  snakes.  It 
is  non-pathogenic  to  warm  blooded 
animals  but  will  kill  a  frog  within  4 
weeks.  It  grows  at  a  temperature 
'from  15°  to  30°  C.  Attempts  have 
been  made  to  show  a  close  relation- 
ship between  the  tubercle  bacillus  of 
cold  blooded  animals  and  that  of 
warm  blooded  animals.  Kuster,  re- 
viewing the  ^work  of  many  investi- 
gators, states  that  spontaneous  tuber- 
culosis may  occur  in  fish,  snakes, 
turtles  and  frogs,  and  that  the  organ- 
ism which  causes  these  diseases  is 
specific  for  cold  blooded  animals  and 
similar  in  many  respects  to  the 
tubercle  bacillus  of  warm  blooded  an- 
imals, but  in  them  they  do  not  pro- 
duce progressive  disease.  The  human, 
bovine  and  avian  tubercle  bacilli 
when  inoculated  into  cold  blooded  an- 
imals can  produce  lesions  which  sim- 
ulate tuberculosis  and  the  bacilli  may 
remain  in  these  lesions  for  a  long 
period  of  time  without  losing  their 
pathogenicity  for  guinea  pigs. 

BACII^Iil    RESEMBIVING  TUBEBd^E 
BACII^IiZ. 

Bacllltis  of  Timothy.  This  organism 
was  isolated  by  Muller  from  timothy 
grass  and  dust  in  haylofts.   Will  grow 


BACTERIOLOGY.  237 

rapidly  on  ag"ar  and  is  of  a  deep  red 
or  dark  yellow  color. 
Baclllxis  Butyricus  (Butter  Bacillus): 
This  organism  was  isolated  by  Petri, 
Korn  and  others  from  milk  and  but- 
ter. It  resembles  the  bacillus  of  tu- 
berculosis in  that  it  is  slightly  acid 
fast  but  can  be  differentiated  cultur- 
ally. It  is  slightly  pathogenic  for 
guinea  pigs  but  not  for  man. 

ZkUSTGABTEN'S  BACIIiZ.US. 

Bacillus  Smegmatis. 

Lustgarten,  in  1884,  discovered  an  acid 
fast  bacillus  in  syphilitic  lesions 
which  he  believed  to  be  the  cause  of 
this  disease.  It  has  since  been  shown 
that  a  bacillus  which  corresponds  in 
its  morphology  but  differing  slightly 
in  certain  staining  peculiarities  to  the 
bacillus  described  by  Lustgarten,  oc- 
curs as  a  harmless  saprophyte  in  the 
normal  smegma  from  the  prepuce  or 
vulva.  The  bacillus  is  a  straight  or 
ciirved  organism  resembling  the  tu- 
bercle bacillus  in  its  morphology.  It 
is  found  lying  singly  or  sometimes  in 
groups  within  the  interior  of  cells 
having  a  round,  oval  or  polygonal 
form  and  apparently  somewhat 
swollen.  It  stains  with  almost  as 
much  difficulty  as  the  tubercle  bacillus, 
but  is  more  easily  decolorized.  A 
method  of  differentiation  was  devised 
by  Pappenheim  (see  stain)  which  de- 
pends upon  the  fact  that  prolonged 
treatment  with  alcohol  and  rosolic 
acid  will  decolorize  the  smegma  bacil- 
lus but  not  the  tubercle  bacillus. 

Attempts  have  been  made,  without  suc- 
cess, to  cultivate  Lustgarten's  bacillus 
on  artificial  media.  The  smegma 
bacillus  has  no  pathogenic  signi- 
ficance. Attempts  to  infect  animals 
have  been  unsuccessful.  It  might  be 
well  to  state  here  «that  although  the 
so  called  smegma  bacillus  and  the 
bacillus  of  Lustgarten  are  almost 
Identical,  the  identity  of  the  two 
bacilli  has  not  been  definitely  estab- 
lished. 

I^EFBOSY  BACZZ^l^US. 

Bacillus  ILeprae, 

The  Bacillus  of  Leprosy  was  discovered 
by  G.  A.  Hansen,  in  1879,  in  the  tis- 
sues of  the  nodular  lesions  of  indi- 


238  BACTERIOLOGY. 


viduals  sufferingr  from  leprosy.  The 
organisms  were  found  lying  in  small 
clumps  intra  and  extracellularly,  as 
well  as  in  the  serum  that  oozed  from 
the  tissue  during  its  removal.  The 
disease  was  present  and  widely  dis- 
tributed long  before  the  Christian 
era,  extending  down  through  the 
Middle  Ages  to  the  present  time.  It 
is  most  common  in  India  and  China. 
It  is  found  in  Norway,  Russia  and 
Iceland;  and  in  the  United  States  in 
Louisianna,  California  and  Minnesota. 
The  bacillus  of  leprosy  is  a  small, 
slender,  acid  fast  rod,  resembling  the 
tubercle  bacillus  in  form,  though 
somewhat  shorter  and  not  so  fre- 
quently curved.  The  rods  have  point- 
ed ends,  and  when  stained  have  the 
unstained  spaces  frequently  seen  in 
the  tubercle  bacilli.  The  organism 
stains  easily  with  the  ordinary  aninine 
dyes,  also  by  Gram's. 
Attempts  to  inoculate  animals  with 
leprosy  have  been  unsuccessful.  Sub- 
cutaneous inoculations  of  cultures 
into  guinea  t>ig"S  have  produced  local 
lesions  which  resemble  the  leprous 
'lesions  in  man.  Duval  states  that  he 
was  able  to  continue  the  growth  on 
later  transfers.  The  bacilli  are  usual- 
ly found  in  large  numbers  especially 
in  the  tubercles  of  the  skin,  in  the 
conjunctiva  and  cornea,  the  mucous 
membrane  of  the  mouth,  gums  and 
larynx,  and  in  the  interstitial  pro- 
cesses of  the  nerves,  testicles,  liver, 
spleen  and  kidneys.  The  bacilli 
nearly  always  lie  within  the  cells  in 
the  old  ceilters  of  infection,  are 
larger  and  often  polynuclear.  Some 
observers  have  claimed  to  have  found 
giant  cells  similar  to  those  of  tuber- 
culosis in  the  nodules.  The  hair  fol- 
licles, sebaceous  sweat  glands,  are 
often  attached.  A  true  caseation  does 
not  occur,  but  ulceration  results. 
In  the  anaesthetic  form  of  leprosy,  the 
bacilli  are  most  comonly  found  in  the 
nerves  and  less  frequently  in  the  skin. 
The  organism  may  also  occur  in  the 
blood,  partly  free  and  partly  within 
the  leucocyte,  particularly  during  the 
febrile  stage,  which  precedes  the 
reaking  out  of  the  tubercles.  The 
acilli  have  also  been  found  in  the 


BACTERIOLOGY.  239 


intestines,  in  the  lungs  and  in  the 
sputum,  but  not  in  the  urine.  ' 

The  contagiousness  of  leprosy  is  far 
less  than  that  of  most  other  bacterial 
diseases.  The  question  of  direct  in- 
heritance of  the  disease  from  the 
mother  to  the  unborn  child  has 
brought  out  a  considerable  difference 
of  opinion.  Many  attempts  have  been 
made  to  infect  healthy  individuals 
with  the  material  containing  the  ba- 
cilli without  conclusive  results.  Arn- 
ing,  although  he  successfully  infected 
a  condemned  criminal  in  the  Sanj^- 
wich  Islands  with  fresh  leprous  tu- 
bercles, did  not  produce  positive  evi- 
dence of  the  transmissibility  of  the 
disease  in  that  way,  as  Swift  pointed 
out  that  the  man  had  other  oppor- 
tunities of  becoming  infected.  A  wide- 
spread idea  before  the  discovery  of 
the  organism  that  the  disease  was 
associated  with  the  constant  eating  of 
dried  fish,  or  certain  kind  of  food,  has 
been  abandoned. 

The  relation  of  leprosy  to  tuberculosis 
is  evidenced  by  their  similarity  in 
many  respects,  and  still  more  so  by 
the  fact  that  leprosy  will  react  both 
locally  and  generally  to  injections  of 
tuberculin  in  the  same  manner  as 
tuberculosis,  but  to  somewhat  less 
extent. 

Cultivation  of  the  leprosy  bacillus  has 
not  met  with  success. 

Clegg,  in  1908,  reported  that  he  had  been 
able  to  cultivate  an  acid  fast  bacillus, 
from  cases  of  leprosy,  in  symbiosis 
with  ameba  and  cholera  vibrio.  By 
heating  a  symbiotic  culture  of  the 
leprosy  bacillus  to  60°  C.  it  was  ob- 
tained in  pure  culture.  From  the  first 
culture,  different  media  were  success- 
fully inoculated.  On  agar,  the  surface 
colonies  are  small  and  brownish. 
Blood  serum  is  liquefied  after  ten 
days;  lactose  is  not  fermented.  Duval 
succeeded  in  obtaining  cultures  by  the 
Clegg  method,  but  in  spite  of  ex- 
tensive work  along  this  line,  the 
opinions  are  still  divided  as  to  the 
specific  nature  of  the  organisms  cul- 
tivated by  these  two  investigators. 


240  BACTERIOLOGY. 

BACII.Z.US   OF  BAT  I^EFBOSY. 

A  disease  occurring-  spontaneously  among 
house  rats  in  Odessa  was  first  ob- 
served by  Stefansky,  characterized  by 
subcutaneous  induration,  swelling  of 
lymphnodes,  falling  out  of  the  hair, 
emaciation  and  sometimes  ulceration. 
In  the  diseased  rats,  under  the  skin 
of  the  abdomen  or  flank,  a  thickened 
area  of  adipose-like  tissue,  though 
more  nodular  and  grey  and  less  shiny 
than  fat,  may  be  found,  within  which 
are  acid  fast  bacilli  resembling  the 
bacillus  leprae.  These  organisms  may 
also  be  found  in  the  lymphnodes  and 
sometimes  in  small  nodules  of  the 
liver  and  lung. 

The  disease  can  be  transmitted  from  rat 
to  rat  experimentally.  The  disease  is 
not  exactly  like  human  leprosy  clin- 
ically. 

PATHOGEKIC     ANAEROBIC  GBOUF 
OF  OBGANISMS. 

BACII^I^US  TETANI. 

The  infectious  nature  of  lockjaw,  or 
tetanus,  was  demonstrated  by  Carlo 
and  Rattone,  in  1884.  Kitasato  by 
'anaerobic  methods  demonstrated  the 
tetanus  bacillus  in  1889,  and  definitely 
solved  the  etiology  of '  the  disease. 
The  bacillus  of  tetanus  is  found  to 
occur  in  the  superficial  layers  of  the 
soil.  The  soil  of  cultivated  and  ma- 
nured fields  is  thickly  sown  with  this 
organism,  probably  because  of  its 
presence  in  the  dejecta  of  some 
doniestic  animals. 

The  organism  is  a  slender  bacillus,  the 
vegetative  forms  of  which  are  slight- 
ly motile  by  reason  of  the  numerous 
peritrichial  flagella.  The  organism 
develops  spores  which  are  character- 
istically located  at  one  end  and  give 
to  it  the  diagnostic  drum  stick  ap- 
pearance. The  vegetative  forms  oc- 
cur chiefiy  in  young  cultures,  which, 
after  24  hours'  incubation,  develop 
into  the  spore  forms,  and  as  the  cul- 
tures grow  old,  the  spores  will  super- 
sede the  vegetative  form.  In  very  old 
cultures  only  spore  forms  and  spores 
are  found.  The  organism  stains  easily 
with  aniline  dyes  and  also  by  Gram. 
The  organism  is  extremely  patho- 
genic, though  viewed  from  the  stand- 


BACTERIOLOGY.  241 

point  of  universal  distribution  of  the 
bacilli  in  nature,  the  infection  is  in- 
frequent. The  spores  of  the  organism 
introduced  into  the  animal  body,  free 
from  toxins,  the  disease  may  not  be 
produced,  by  reason  of  their  suscep- 
tibility to  destruction  by  phagocytosis 
and  to  other  protective  agents  before 
vegetative  forms  can  develop  and  the 
toxin  formed.  Tetanus  will,  however, 
develop  in  animals  if  introduced  into 
deep,  lacerated  wounds  in  which  there 
has  been  considerable  tissue  destruc- 
tion. Common  pus  cocci  or  other 
more  harmless  parasites  may  aid  in 
furnishing  a  suitable  media  for  the 
growth  of  the  tetanus  bacillus.  There 
is  an  incubation  period  of  from  5  to 
7  days  in  acute  cases  to  from  4  to  5 
weeks  in  chronic  ones.  Guinea  pigs 
inoculated  with  the  organism  sufCer 
in  from  1  to  3  days  from  a  rigidity 
of  the  muscles  nearest  the  point  of 
infection,  which  rapidly  spreads  to 
other  parts  of  the  body  and  is 
followed  by  death  in  4  or  5  days  after 
the  time  of  injection.  Autopsies  ot 
animals  or  human  beings,  dead  of 
tetanus,  do  not  present  marked  lesions. 
The  point  of  infection  may  be  triffling 
in  appearance.  The  organs  show  no 
pathological  changes  except  a  general 
moderate  congestion.  The  bacilli  are 
infrequently  found  at  the  point  of  in- 
fection and  have  rarely  been  demon- 
strated in  the  blood  or  viscera.  Tiz- 
zoni  and  Creite  have  succeeded  in  cul- 
tivating the  tetanus  bacillus  from  the 
spleen  and  heart's  blood  of  infected 
human  beings.  The  pathogenicity  of 
the  organism  is  dependent  upon  the 
soluble  toxin  which  it  produces.  It 
is  one  of  the  most  powerful  poisons 
known.  Filtrates  of  broth  cultures  in 
quantities  of  0.000.005  cc.  will  often 
prove  fatal  to  mice  of  10  gm.  weight. 
Different  species  of  animals  show 
great  variation  in  susceptibility. 
Human  beings  and  horses  are  prob- 
ably the  most  susceptible  species  in 
proportion  to  their  body  weight.  The 
susceptibilty  of  the  horse  calculated 
for  grams  of  body  weight  is  twelve 
times  that  of  a  mouse.  The  hen  Is 
extremely  resistant,  in  fact  200,000 
times  more  resistant  than  the  mouse. 
The    toxin    injected  subcutaneously 


242 


BACTERIOLOGY. 


first  produces  spasms  in  the  muscles 
nearest  the  point  of  inoculation.  In- 
travenous inoculation  usually  results 
in  a  general  spasm  of  all  the  muscles. 
The  action  of  the  tetanus  toxin  is 
upon  the  central  nervous  system.  The 
manner  in  which  the  toxin  reaches  the 
central  nervous  system  is  by  way  of 
the  motor  nerve.  The  toxin  Injected 
into  the  circulation  will  reach  all  the 
motor  nerve  endings  simultaneously, 
producing  a  general  tetanus.  The 
poison  cannot  pass  directly  into  the 
central  nervous  system  but  must 
follow  the  route  of  nerve  tracts  (see 
also  antitetanic  serum). 
The  bacillus  of  tetanus  is  an  obligative 
anaerobe  and  if  cultivated  strictly 
under  these  conditions,  it  will  grow 
readily  upon  meat  infusion  broth 
which  becomes  clouded  in  24  to  36 
hours.  Upon  meat  infusion  gelatin, 
there  is  slow  liquefaction.  On  agar, 
at  the  end  of  48  hours,  compact  col- 
onies not  unlike  subtilis  colonies  make 
their  appearance.  In  agar  stabs,  the 
growth  appears  as  fine,  radiating  pro- 
cesses grown  from  the  central  stab. 
Milk  is  a  favorable  media  and  is  not 
*  changed.  On  potato,  a  hardly  visible 
growth  appears.  The  media  may  be 
rendered  more  favorable  for  growth 
by  the  addition  of  a  1  or  2%  of 
glucose,  maltose  or  sodium  formate. 
The  vegetative  forms  of  the  bacillus 
are  not  more  resistant  against  heat  or 
chemical  agents  than  the  vegetative 
forms  of  other  microorganisms.  The 
spores,  however,  will  resist  dry  heat 
at  80°  C.  for  1  hour,  live  steam  for 
about  5  minutes.  They  are  killed  by 
a  5%  carbolic  acid  solution  in  12  to 
15  hours;  1%  of  bichloride  of  mer- 
cury in  2  to  3  hours.  Direct  sunlight 
will  diminish  their  virulence  and 
ultimately  destroy  them. 

BACIIiI^nS    OF  SYMPTOMATIC 
ANTHRAX. 

An  infectious  disease  occurring  among 
sheep,  cattle  and  goats,  spoken  of  as 
"Quarter-evil"  or  "Blackleg"  is  often 
confused  with  true  anthrax  by  reason 
of  a  slight  similarity  in  clinical  symp- 
toms. It  is,  however,  caused  by  a 
very  different  microorganism  found 
widely  distributed   in  the  soil,  from 


BACTERIOLOGY. 


243 


which  the  infection  is  generally  ob- 
tained. The  bacillus  of  symptomatic 
anthrax  is  a  spore  bearing  bacillus  of 
drum  stick  shape,  or  spindle  shape, 
and  is  anaerobic.  It  was  first  ob- 
tained in  pure  cultures  by  Kitasato. 
It  is  usually  seen  singly  and  never 
forms  chains.  In  the  vegetative  form 
the  organism  is  motile,  but  soon  loses 
this  power  on  account  of  the  oxygen 
to  which  it  is  exposed.  It  shows  well 
defined  flagella  and  develops  spores. 
The  organism  may  be  demonstrated 
by  the  aid  of  microscope  in  the  blood 
without  staining,  if  done  soon  after 
death.  It  stains  easily  by  the  sim- 
ple aniline  dyes  or  by  Gram's. 

The  bacilli  are  pathogenic  for  cattle, 
sheep  and  goats.  Most  cases  appear 
among  cattle.  Guinea  pigs  are  very 
susceptible.  Horses,  little  suscepti- 
ble; dogs,  cats,  rabbits  and  birds  are 
immune.  Man  appears  to  be  abso- 
lutely immune.  Infection  takes  place 
through  abrasions  or  wounds,  and  de- 
pends to  some  extent  upon  the  degree 
of  virulence,  which  varies  greatly  in 
this  organism.  A  soft,  puffy,  swelling 
appears  at  the  point  of  entrance  at 
from  -Z  to  24  hours  after  the  inocu- 
lation. This  area  is  found  to  be 
emphysematous,  which  spreads  rap- 
idly, often  reaching  the  abdomen  and 
chest  in  a  day.  It  is  accompanied 
with  high  fever  and  extreme  general 
prostration.  Death  usually  results 
within  3  to  4  days.  At  autopsy,  the 
swollen  area  is  found  infiltrated  with 
a  thick  blood  tinged  and  foamy  exu- 
date. The  subcutaneous  tissues  and 
muscles  are  edematous  and  filled  with 
gas.  The  organs  show  parenchy- 
matous degeneration  and  hemorraghic 
areas.  The  organisms  are  found  in 
enormous  numbers  in  the  area  sur- 
rounding a  central  focus,  immediately 
after  death,  but  very  few  are  found 
in  the  blood  and  internal  organs.  The 
unburied  carcasses  become  bloated 
with  gas  and  the  organs  filled  with 
bubbles,  by  reason  of  the  general 
distribution  of  the  bacilli. 

A  soluble  toxin  is  produced  by  the  or- 
ganism in  considerable  quantities  in 
broth  containing  blood  or  albuminous 
animal  fiuids,  but  not  to  any  extent 
in  ordinary  broth.    The  toxin  is  quite 


244  BACTERIOLOGY. 

resistant  to  heat,  but  deteriorates  on 
exposure  to  air. 


Arloing  actively  immunized  cattle  by 
subcutaneous  inoculation  with  vac- 
cines prepared  from  the  tissue  of  in- 
fected animals.  Two  vaccines  are 
prepared;  No.  1  is  made  from  the 
juice  of  infected  meat,  which  is  dried 
and  heated  to  100"  C.  for  six  hours. 
No.  2  is  also  made  from  the  juice  of 
infected  meat  heated  to  90°  C.  for 
six  hours.  No.  1  is  injected  in  quan- 
tities of  0.01  to  0.02  cc,  emulsified  in 
sterile  salt  solution,  near  the  end  of 
the  animal  to  be  protected.  The  same 
quantity  of  No.  2  is  injected  in  the 
same  way  after  14  days. 


Kitt  prepared  a  vaccine  from  the  powd- 
ered meat  which  was  heated  to  94°  C. 
for  six  hours,  which  has  been  largely 
used  in  America.  A  passive  immuni- 
zation with  the  serum  of  actively  im- 
munized sheep  has  been  used  in  com- 
bination with  the  methods  of  active 
immunization. 


The  organism  grows  readily  under  an- 
aerobic conditions  upon  the  ordinary 
media,  which  may  be  rendered  more 
favorable  by  the  addition  of  glucose 
glycerine  or  nutrose.  The  organism 
will  grow  equally  well  on  slightly 
alkaline  or  slightly  acid  medium  and 
is  an  active  gas  producer.  On  agar 
plates  the  colonies  appear  round  with 
a  compact,  slightly  granular  center 
from  which  a  thin  zone  is  given  off. 
This  zone  will  under  the  microscope 
appear  as  a  tangle  of  fine  threads.  In 
agar  stabs,  the  growth  appears  with- 
in 18  hours  and  spreads  into  the 
media  as  a  diffused  fine  cloud.  Gas 
bubbles  are  formed  which  later  in- 
crease to  such  extent  as  to  cause  ex- 
tensive splitting  of  the  medium.  On 
gelatin  plates,  round  or  oval  colonies 
with  a  compact  center,  about  which 
are  fine,  radiating  filaments,  make 
their  appearance  in  about  24  hours. 
The  gelatin  is  liquefied.  In  gelatin 
stab  cultures  the  growth  Is  less  rapid, 
but  is  similar  to  the  growth  in  agar 
stabs. 


BACTERIOLOGY.  245 


BACni^US  OF  MAI^IGKANT  EDEMA. 
(Bacillus  Oedamatis  Maligul). 

Pasteur  in  1877  described  an  organism 
which  he  isolated  under  anaerobic 
conditions  in  impure  cultures  from 
the  tissues  of  guinea  pigs  and  rabbits 
which  he  had  inoculated  with  putrefy- 
ing animal  tissues.  He  named  it 
**Vibrion  septique."  Koch,  in  1881,  sug- 
gested the  term  "bacillus  of  malignant 
edema,"  by  reason  of  the  fact  that  the 
organism  did  not  produce  a  true  sep- 
ticemia. The  organism  was  later 
found  to  occur  in  garden  soil  and  in 
dust  by  Gaffky.  The  bacillus  of  ma- 
lignant edema  is  a  long,  slender  rod, 
resembling  somewhat  the  bacillus  of 
anthrax.  It  occurs  singly,  but  fre- 
quently appears  in  long  threads  with 
irregular  subdivisions;  or  without 
subdivision  as  long  homogeneous  fila- 
ment. The  organism  is  motile,  pos- 
sessing .numerous  laterally  placed 
flagella.  Motility  is  often  absent. 
Oval  spores  are  found  irregularly 
placed  in  the  center  or  slightly  nearer 
the  end  and  cause  a  bulging  of  the 
body.  It  stains  readily  by  the  usual 
aniline  dyes  but  is  decolorized  by 
Gram. 

The  organism  seems  to  be  pathogenic 
for  all  animals.  Subcutaneous  inocu- 
lation of  pure  cultures  produces  an 
acute  edematous  inflammation  at  the 
point  of  inoculation  within  24  to  36 
hours.  This  edema  spreads  through- 
out the  subcuticular  and  deeper  layers 
and  consists  of  a  thin  fluid  which  is 
slightly  bloody.  The  lymphnodes  be- 
come enlarged  and  hemorrhagic.  Gas 
is  formed  causing  subcutaneous  em- 
physema. The  toxemia  is  general,  and 
in  small  animals  the  disease  is  usual- 
ly fatal.  At  autopsy  the  organisms 
are  found  in  the  edema  about  the 
local  lesion.  The  organism  is  not 
found  in  the  blood  or  internal  organs 
shortly  after  death,  though  later  they 
may  become  distributed  throughout 
the  body.  The  internal  organs  show 
parenchymatous  degeneration  with  oc- 
casional hemorrhages. 

Infection  with  malignant  edema  Is  rare. 
It  has  been  seen  in  horses,  in  cattle 
and  in  sheep.  Infection  generally 
takes  place  after  tramua,  and  in  man 


246  BACTERIOLOGY. 


secondarily,  after  compound  frac- 
tures, or  upon  the  site  of  suppurating 
wounds. 

A  recovery  from  the  infection  will  pro- 
duce immunity.  Immunity  may  also 
be  produced  by  the  injection  of  solu- 
ble toxin  in  bacteria-free  filtrates  of 
the  organism.  Very  small  amount  of 
this  toxin  is  capable  of  killing  guinea 
pigs.  Immunity  can  also  be  produced 
by  injection  with  the  toxic  filtered 
sera  of  animals  dead  from  the  disease. 

The  bacillus  of  malignant  edema  may 
be  cultivated  under  strict  anaerobic 
conditions  upon  any  of  the  ordinary 
media.  The  addition  of  .glucose  to  the 
media  will  favor  its  growth.  Gases 
are  produced  through  proteid  cleav- 
age. On  gelatin  plates,  small,  grey, 
spherical  colonies,  with  microscopic 
radial  filaments,  appear  in  about  3 
days.  The  gelatin  is  liquefied.  In  the 
gelatin  stab,  growth  appears  along 
the  entire  stab  to  within  a  short  dis- 
tance of  the  surface.  Processes  de- 
velop laterally  with  a  formation  of 
gas.  In  agar,  the  growth  appears  as 
a  white  line  along  the  entire  length 
of  the  stab,  which  takes  on  a  lateral 
*  cloud-like  extension,  and  in  the  pres- 
ence of  sugar,  bubbles  will  be  formed 
throughout  the  medium.  In  broth, 
there  is  general  clouding  and  a  gran- 
ular precipitate.  Milk  is  slowly  co- 
agulated. 

BACIXiI^irS  AEBOGENES 
CAFSUIiATUS. 

The  Bacillus  Aerogrenes  CapsulatTiB  is 

found  in  soil,  dust  and  brackish  water 
and  in  the  intestine  of  human  beings 
and  animals.  They  were  first  ob- 
served by  Welch  in  1892,  having  ob- 
tained it  during  autopsy  from  the 
blood  of  a  case  of  ruptured  aortic 
aneurysm.  His  attention  was  called 
to  the  blood  by  reason  of  air  bubbles 
appearing  throughout  the  vessels. 
The  organism  appears  as  a  straight  rod 
of  varying  length,  and  somewhat 
thicker  but  not  unlike  the  anthrax 
bacillus.  On  rare  occasions  the  or- 
ganism may  be  slightly  curved,  and 
still  rarer  it  may  be  so  short  as  to 
appear  almost  coccoid.  The  organism 
generally  appears  singly  with  rounded 
ends,  but  may  appear  as  short  chains 


BACTERIOLOGY.  247 


when  the  ends  are  almost  square.  The 
chain  formation  takes  place  in  the 
blood.  Long  chains  are  never  seen  in 
artificial  culture  media  which  dis- 
tinguishes the  organism  from  the 
bacillus  of  anthrax.  Spores  are  seen 
in  blood  serum  cultures,  rarely  upon 
plain  agar  and  never  in  the  animal 
body.  They  are  oval  in  form  and 
may  be  centrally  or  polarly  located. 
It  is  non  motile  and  enclosed  by  a 
capsule  which  cannot  always  be 
demonstrated.  The  capsules  are  best 
seen  when  preparations  are  made 
from  animal  fluid,  though  they  may 
be  seen  in  specimens  grown  upon  arti- 
ficial culture  media.  It  is  stained 
readily  by  the  ordinary  aniline  dyes 
and  by  Gram,  if  taken  from  tissues. 
If  taken  from  artificial  culture  media, 
they  are  partially  decolorized  by  Gram 
by  reason  of  involution  forms. 
The  bacillus  is  highly  pathogenic  for 
guinea  pigs  but  very  slightly  so  for 
rabbits.  The  virulence  varies  with 
the  strain.  In  general,  its  patho- 
genicity for  laboratory  animals  is 
slight.  The  bacillus  has  been  isolated 
from  numerous  cases  of  emphy- 
sematous gangrene  in  man,  which  is 
characterized  by  a  rapidly  necrotizing 
inflammation  accompanied  by  subcu- 
tanecrus  emphysema.  Infection,  when 
it  takes  place,  follows  trauma,  especi- 
ally compound  fractures.  The  organ- 
ism has  also  been  found  in  the  uterus 
in  puerperal  infection.  It  has  also 
been  found  in  infectious  processes  of 
gastrointestinal  and  biliary  tracts,  the 
lungs,  the  pleura  and  the  meninges. 
*The  organism  may  be  isolated  from 
mixed  cultures  by  animal  inoculation. 
About  1  cc.  of  the  suspected  material 
is  emulsified  with  about  5  cc.  of  sterile 
salt  solution  and  filtered  through 
sterile  paper.  1  to  2  cc.  of  this  sus- 
pension is  injected  into  the  ear  vein 
of  a  rabbit.  The  rabbit  is  then  killed 
and  placed  in  the  incubator  for  five 
to  eight  hours,  at  the  end  of  which 
times  the  carcass  will  be  found  to 
be  distended  with  gas.  Autopsy  will 
show  gas  to  be  distributed  as  bubbles 
'  throughout  the  organs.  Prom  these 
bubbles,  the  organism  may  be  taken 
for  indentification  or  culture.  The  or- 
ganism is  grown  under  obligatory  an- 


248  BACTERIOLOGY. 

aerobic  conditions  upon  any  of  the 
usual  media  of  a  neutral  or  slightly 
alkaline  reaction.  The  addition  of  glu- 
cose or  lactose  will  favor  the  growth. 
Upon  plate  cultures  a  flat  grayish, 
translucent,  round  colony  with  slight- 
ly irregular  and  fringed  margin  ap- 
pears within  24  hours.  Gelatin  is 
slowly  liquefied,  but  occasionally 
liquefaction  does  not  occur.  In  sugar- 
ed agar  stabs  or  slants,  there  is  a 
rapid  formation  of  gas  which  is  con- 
sidered of  diagnostic  value.  In  broth, 
there  is  general  clouding,  and  with- 
in 40  hours  a  heavy,  white  flocculent 
sediment  is  formed.  Froth  appears 
upon  the  surface  of  the  broth  tubes, 
if  undisturbed,  due  to  the  formation 
of  gas.  On  potato,  the  growth  is 
scanty.  On  coagulated  blood  serum, 
the  growth  is  heavy  with  slight  pep- 
tonization of  the  medium.  Milk  is 
rapidly  coagulated  and  rapidly  acidi- 
fied.   Gas  is  formed. 

THE  BACIUUS  ENTEBITIDIS 
SFOROGENES. 

This  organism  closely  resembles  the 
bacillus  aerogenes  capsulatus.  It  is 
•  a  spore  bearing  organism  usually 
present  in  the  intestinal  tract  of  man. 
It  is  found  in  sewage,  milk,  dust  and 
food  stuffs,  such  as  wheat,  oatmeal, 
rice,  etc.  Cline  believed  that  it  pro- 
duced diarrhea  when  taken  in  milk. 
This  fact  is  disputed  by  many. 

BACII^I^US  BOTUIiINUS. 

The  Bacillus  Botulinus  was  discov- 
ered by  van  Ermengen,  in  1896,  who 
isolated  the  organism  from  a  ham, 
the  eating  of  which  had  caused  dis- 
ease in  a  number  of  persons.  He 
found  the  bacilli  within  the  muscle 
fibers  of  the  ham  in  great  numbers 
and  was  also  able  to  cultivate  the 
same  microorganism  from  the  stomach 
and  spleen  of  one  of  the  individuals 
who  ^ied  from  the  infection.  The 
bacillus  is  a  large,  straight  rod  with 
rounded  ends,  appearing  singly  or 
grouped  in  very  short  chains.  It  is 
slightly  motile  and  develops  oval 
spores  which  are  situated  near  the 
end  of  the  bacillus,  on  rare  occasions 
at  the  middle.  It  is  stained  readily 
by  the  ordinary  aniline  dyes  also  by 


BACTERIOLOGY.  249 

Gram,  if  care  be  taken  that  there  is 
not  over  decolorization  with  the  al- 
cohol. 

Ingestion  of  meat  infected  with  this 
org-anism  produces  the  botulism  or 
allantiasis  in  man.  After  a  period  of 
incubation  of  from  24  to  48  hours,  the 
symptoms  are:  chilliness,  trembling, 
giddiness,  followed  by  headache,  occa- 
sionally by  vomiting.  The  chief  diag- 
nostic symptoms  are  due  to  the  tox:ic 
interference  with  the  cranial  nerves, 
producing  loss  of  accommodation, 
dilated  pupils,  ptosis,  aphonia  and 
dysphagia,  etc.  Fever  is  usually  ab- 
sent. Consciousness  is  rarely  lost. 
These  symptoms  difCer  from  the  meat 
poisonings  caused  by  other  micro- 
organisms. 

Animals  inoculated  with  living  cultures 
or  toxins  will  show  the  symptoms  in- 
dicated above.  Guinea  pigs  seem  to 
be  the  most  susceptible.  They  are 
killed  by  injections  of  minute  quan- 
tities of  the  toxin.  Before  death, 
which  occurs  within  24  to  36  hours, 
general  motor  paralysis,  dyspnea,  and 
hypersecretion  of  mucus  from  nose 
and  mouth  may  occur. 

Autopsies  show  a  general  hyperemia  of 
the  organs  with  much  parenchyma- 
tous degeneration  and  many  minute 
hemorrhages. 

The  poisoning  occuring  in  man  is  due 
to  the  toxins  that  have  been  formed 
by  the  bacillus  in  the  ingested  meat. 
It  has  been  shown  that  little  or  no 
toxin  is  produced  by  the  bacillus  after 
it  has  been  introduced  into  the  ani- 
mal body.  It  produces  the  disease  by 
the  absorption  of  the  toxin  that  is 
secreted  by  the  organisms.  This  toxin 
Is  active  not  only  when  injected  sub- 
cutaneously,  but  also  when  intro- 
duced through  the  gastrointestinal 
canal.  A  specific  antitoxin  has  been 
produced  by  Kempner. 

The  organism  is  easily  cultivated  under 
strict  anaerobic  conditions  in  the  or- 
dinary culture  media  of  a  neutral  or 
moderately  alkaline  reaction  and  at 
a  temperature  not  exceeding  35"  C. 
On  gelatin,  the  growth  is  rapid  and 
abundant  with  the  formation  of  gas 
and  rapid  liquefaction.  On  glucose 
gelatin  plates,  the  colonies  appear  as 
round,    yellowish,    transparent  spots 


250  BACTERIOLOGY. 


surrounded  by  a  zone  of  liquefaction. 
On  agar  plates,  the  colonies  are  yel- 
lowish, opalescent  and  round  in  shape, 
with  a  finely  fringed  border.  Stab 
cultures  in  glucose  agar  produce  a 
thin  white  growth  along  the  line  of 
stab  which  does  not  reach  the  surface 
of  the  medium.  The  medium  is  split 
by  the  formation  of  gas.  Milk  is  not 
coagulated.  Disaccharides  and  poly- 
saccharides are  not  fermented.  The 
gas  that  is  formed  in  cultures,  chiefly 
hydrogen  and  methane. 

KEMOBBKAGIC  SEPTICEMIA  GROUP 
OP  ORGANISMS. 
BACTERIUM  PESTIS. 
(Bacillus  of  Bubonic  Plargnie). 

The  bacterium  of  bubonic  plague  was 
described  by  Yerdin  and  Kitasato  in 
1893,  independently  of  each  other,  and 
is  now  recognized  as  the  etiological 
factor  in  bubonic  plague,  epidemics 
of  which  have  been  recognized  since 
the  second  century.  About  half  the 
population  of  the  Roman  Empire  died 
in  the  sixth  century,  and  during  the 
fpurteenth  century  an  epidemic  (The 
Black  Death)  swept  over  Europe  and 
killed  about  twenty-five  million  peo- 
ple. About  two  million  died  of  this 
disease  in  India  during  1901  to  1904. 
Smaller  epidemics  have  appeared  in 
numerous  parts  of  the  world,  as 
China,  Egypt  and  South  Africa. 

The  organism  is  a  short,  thick  bacillus 
with  well-rounded  ends.  It  is  non- 
motile,  and  appears  singly,  in  pairs 
and  occasionally  in  short  chains.  The 
organism  shows  distinct  polar  stain. 
The  size  of  the  organism  varies,  and 
in  old  cultures  involution  forms  may 
appear  either  as  club-shaped  diph- 
theria-like bacilli  or  as  swollen 
coccoid  forms.  The  involution  forms 
are  of  diagnostic  importance  by  rea- 
son of  their  irregularity,  and  seem  to 
be  more  numerous  in  artificial  media. 
There  are  no  spores  present,  and  cer- 
tain observers  have  demonstrated  a 
erelatinous  capsule.  Occasionally, 
branched  forms  may  be  observed.  It 
stains  easily  with  aniline  dyes,  par- 
ticularly, at  ^he  poles.  It  is  Gram 
negative. 


BACTERIOLOGY.  251 


The  organism  is  extremely  pathogenic 
for  rats,  mice,  guinea  pigs,  rabbits 
and  monkeys.  Even  insects  die  from 
the  infection.  Among  animals,  the 
disease  has  been  found  chiefly  in  rats 
and  squirrels.  Dogs  may  occasionally 
become  infected.  Two  distinct  types 
are  observed  clinically,  depending 
upon  the  mode  of  infection,  which 
takes  place  by  the  entrance  of  organ- 
ism through  the  skin  or  by  the  res- 
piratory tract.  In  the  cutaneous 
infection,  which  may  take  place 
through  the  most  minute  lesion,  the 
disease  is  first  localized  in  the  nearest 
lymphnodes,  and  from  these  primary 
dwellings  the  bacilli  may  enter  the 
blood  and  produce  secondary  foci.  If 
entrance  has  taken  place  through  the 
respiratory  tract,  a  pneumonia  is  pro- 
duced which  usually  proves  fatal 
within  four  or  five  days.  Cardiac  de- 
pression is  a  very  characteristic 
symptom  of  systemic  infection.  The 
diagnosis  may  be  made  during  life  by 
finding  the  bacilli  in  the  aspirated 
fluid  from  a  bubo,  or  from  the 
sputum.  Identification  is  made  mor- 
phologically, culturally,  animal  inocu- 
lation and  agglutination  tests. 

At  autopsy,  in  man,  the  bacilli  are 
found  in  the  primary  lesion  in  the 
blood  and  in  the  spleen,  the  liver  and 
lymphatics.  Hemorrhages  into  the 
serous  cavities  may  be  found.  The 
pneumonic  type  is  usually  of  the 
bronchopneumonic  variety,  with  ex- 
tensive swelling  of  the  bronchial 
lymphnodes.  When  the  disease  has 
been  prolonged,  tubercle-like  foci  may 
be  found  in  the  spleen,  liver  and  lung. 

The  typical  lesions  found  in  rats,  who ' 
play  an  important  role  in  the  spread 
of  the  disease,  becoming  infected 
from  the  cadavers  of  plague  victims 
or  the  eating  of  the  bodies  of  rats, 
dead  from  the  disease,  are  engorge- 
ment of  the  subcutanous  vessels  and 
a  pink  colorization  of  the  muscles. 
The  bubo  when  present  is  suflacient 
for  diagnosis.  The  area  siirrounding 
the  buboes  are  markedly  injected  and 
sometimes  hemorrhagic.  A  pleural  in- 
fusion is  present.  The  liver  has 
undergone  a  fatty  change.  The  spleen 
is  large,  friable  and  often  presents 
pinpoint  granules  on  the  surface. 


252  BACTERIOLOGY. 


The  systemic  symptoms  are  due  to  the 
absorption  of  the  toxin,  which  are  of 
the  endotoxin  and  also  of  a  true 
soluble  toxin  variety. 

A  single  attack  will  immunize.  The 
antibodies  developed  are  agglutinins, 
bacteriolytic  and  possibly  antitoxin. 
The  agglutinins  are  of  importance  in 
diagnosis.  Active  immunization  is 
accomplished  by  the  inoculation  of 
the  whole  dead  bacteria.  The  serum 
of  immunized  animals  has  been  tried 
as  a  therapeutic  agent  and  gives  en- 
couraging results  when  administered 
early. 

The  bacillus  is  isolated  in  pure  culture 
from  the  lesion  during  life  or  at 
autopsy.  The  organism  grows  read- 
ily, best  upon  meat  infusion  of  a 
neutral  or  moderate  alkaline  reaction 
and  at  a  temperature  of  30*  C.  It 
will  grow  at  temperatures  ranging 
from  20*'  to  38°  C.  On  agar,  minute 
colonies  with  a  compact  center  sur- 
rounded by  an  irregular,  indented, 
granular  margin  appears  within  24 
hours.  On  gelatin,  colonies  like  those 
upon  agar  appear  after  two  or  three 
days.  It  is  not.  liquefied.  In  bouillon, 
t^he  organism  grows  slowly,  sinking 
to  the  bottom  or  adhering  to  the 
sides  of  the  tube  as  granular  deposit. 
Occasionally  a  delicate  pellicle  is 
formed.  Milk  is  not  coagulated.  In 
litmus  milk,  there  is  slight  acid 
formation.  No  indol  is  formed  upon 
peptone. 

The  organisms  are  eliminated  in  the 
exudates  from  surrounding  buboes 
and  from  the  sputum  in  the  pneu- 
monic type  and  are  present  through- 
out the  body  after  death;  therefore, 
the  dead  bodies  of  human  beings  and 
of  rats  are  sources  of  infection  for 
other  rats,  which  become  chronic  car- 
riers of  the  disease;  and  even  though 
showing  no  symptoms  themselves, 
they  must  necessarily  be  important 
factors  in  the  maintenance  and 
spread  of  the  disease. 

If  in  a  dark  and  moist  environment,  the 
organism  may  live  outside  of  the 
body  for  months  and  even  years.  In 
cadavers,  they  may  live  for  weeks 
and  months,  if  protected  from  dry- 
ness. Complete  drying  will  kill  the 
organism  in  two  or  three  days,  and 


BACTERIOLOGY.  253 


dried  artificially,  they  may  be  killed 
within  four  or  five  hours.  A  dry  heat 
of  100°  C  will  kill  the  organism  in  one 
hour,  boiling  the  organism  will  kill  it 
in  a  few  minutes.  They  are  very  re- 
sistant to  cold.  Direct  sunlight 
destroys  them  within  four  or  five 
hours.  They  are  not  very  resistant 
to  antiseptics. 
Prophylaxis  consists  in  isolating  in- 
fected animals,  followed  by  thorough 
disinfection,  involving  even  the  kill- 
ing of  fleas  and  the  destruction  of 
rats,  squirrels  or  other  animals  which 
may  serve  as  carriers.  Haffkine's 
vaccination  method  has  also  been 
shown  to  be  a  valuable  prophylactic 
measure. 

BACTEBIUM  TUI^ABEINSZ!. 

McOoy  has  described  a  disease  which 
occurs  in  the  California  ground  squir- 
rel which  closely  resembles,  so  far  as 
the  lesions  are  concerned,  the  plague 
infection  of  these  animals.  McCoy 
was  able  to  transmit  plague-like 
lesions  in  most  animals  inoculated 
with  infected  material,  and  later,  in 
1912,  McCoy  and  Chapman  isolated 
the  bacterium  on  an  egg  medium. 
The  organism  is  small  and  often  cap- 
sulated,  staining  poorly  with  methy- 
lene blue,  better  with  carbo  fuchsin 
or  gentian  violet. 

BACTERIUM  AVISEFTICUS. 
(Bacillus  of  Chicken  Cholera). 

The  bacillus  of  chicken  cholera  was 
discovered  in  the  blood  of  infected 
animals  by  Pasteur  in  1880.  This 
•  disease  is  widely  prevalent  in  chick- 
ens, ducks,  geese  and  a  large  variety 
of  smaller  birds.  The  organism  is  a 
short  non-motile  rod,  staining  easily 
with  analine  dyes.  It  is  decoLorized 
by  Gram.  It  often  appears  as  a 
diplococcus  by  reason  of  marked  polar 
staining  qualities.  Spores  are  not 
formed.  Occasionally  vacuolated 
forms,  not  unlike  those  of  tl^e  bacillus 
pestis,  occur.  The  infection  of  birds 
is  extremely  acute,  accompanied  by 
diarrhea,  often  bloody  stools,  great 
exhaustion,  drowsiness,  and  ending 
fatally  within  a  few  days.  Autopsy 
shows  hemorrhagic  infiltration  of  the 


254  BACTERIOLOGY. 


intestinal  mucosa,  enlarg-ement  of  the 
liver  and  spleen  and  frequently  a 
bronchopneumonia. 

The  bacilli  may  be  found  in  the  blood, 
in  the  organs  and  in  the  exudates,  if 
present,  and  in  large  numbers  in  the 
dejecta.  It  grows  well  upon  the  ordi- 
nary culture  media  at  a  temperature 
of  25°  to  40°  C.  Broth  is  clouded  uni- 
formly with  a  later  formation  of  a 
pellicle.  Upon  agar,  minute  white  or 
yellowish,  first  transparent,  later, 
opaque  colonies  appear  within  24  to 
48  hours.  No  liquefaction  of  gelatin. 
No  coagulation  of  milk.  No  gas  is 
formed  in  sugar  broth,  which,  how- 
ever, becomes  acid,  and  in  peptone, 
indol  is  formed. 

Infection  takes  place  probably  through 
food  and  water  contaminated  by  dis- 
charges of  diseased  birds.  The  feed- 
ing or  subcutaneous  inoculation  with 
cultures,  even  in  the  most  minute 
dose,  will  produce  a  quickly  develop- 
ing septicemia  which  is  uniformly 
f3.tal. 

The  bacillus  is  extremely  pathogenic 
for  rabbits,  if  subcutaneous  inocula- 
tions are  made;  less  so  for  hogs, 
sheep  and  horses.  The  disease  does 
not  follow  the  ingestion  of  infected 
material  by  these  animals. 

BACTERIUM  SUIS£FTICUS. 
(Bacillus  of  Swine  Plague). 

The  bacillus  of  swine  plague  causes 
an  epidemic  disease  among  hogs  char- 
acterized by  a  bronchopneumonia, 
which  is  followed  by  a  general  sep- 
ticemia. A  pleural  exudate  of  a  sero- 
sanguineous  nature,  together  with 
enlargements  of  the  bronchial  lymph 
glands,  the  liver  and  the  spleen,  is 
frequently  found.  The  gastrointesti- 
nal .  tract  is  rarely  involved.  At 
autopsy  a  nonmotile  Gram  negative 
bacillus,  almost  identical  with  the 
bacillus  of  chicken  cholera,  may  be 
found  in  the  lungs,  in  the  exudates, 
in  the  liver,  spleen  and  in  the  blood. 
The  disease  is  seldom  acijLte,  but  is 
almost  uniformly  fatal  in  young  pigs. 

Spontaneous  infection  usually  occurs 
by  inhalation.  Experimentally  the 
disease  has  been  produced  by  sub- 
cutaneous inoculation.     Mice,  guinea 


BA<5tERIOLOGY.  255 


pigs  and  rabbits  inoculated  subcu- 
taneously  with  small  doses  of  the 
organism  are  killed  within  four  or 
five  days.  Active  and  passive  im- 
munization has  been  successful. 
Kitt  and  Mayr  have  shown  that  the 
serum  of  horses  immunized  with 
chicken  cholera  would  sometimes  pro- 
tect against  bacillus  suisepticus. 

BACTERIUM  BOVISZSFTIUM. 

The  bacterium  boviseptium  produces  a 
disease  and  affects  a  wide  variety  of 
domestic  and  wild  animals.  It  has 
been  reported  from  many  portions  of 
North  America,  some  sections  of 
South  America  and  many  European 
countries,  and  is  known  as  Corn  Stalk 
disease,  Buffalo  disease  and  pneu- 
monenteritis,  etc.  The  domestic  ani- 
mals most  commonly  affected  are 
cattle,  sheep,  horses  and  goats.  The 
onset  of  the  disease  is  sudden  and 
the  case  acute.  It  does  not  spread 
from  herd  to  herd,  but  appears  in 
isolated  outbreaks.  Body  infection 
probably  occurs  by  inoculation.  The 
characteristic  lesions  found  at  au- 
topsy are  hemorrhages,  which  occur 
subcutaneously  under  the  mucus 
membrane  or  under  the  serous  mem- 
brane and  also  in  the  lymph  glands. 
The  lesions  produced  by  this  bac- 
terium indicate  a  general  distribution 
through  the  body. 

The  mortality  ranges  from  50  to  80  per 
cent.  The  acute  and  rapidly  fatal 
cases,  where  the  autopsy  shows  only 
trifling  lesions,  would  indicate  the 
formation  of  active  toxins. 

Very  little  is  known  concerning  the 
elimination  of  this  organism  from  the 
diseased  body,  but  isolation  and  dis- 
infection are  to  be  recommended  on 
general  principles. 

The  disease  resembles  anthrax  and 
symptomatic  anthrax  in  some  of  its 
characteristics.  It  may  be  readily 
differentiated  from  either  of  these 
diseases  by  microscopic  examination. 

The  organism  resembles  the  bacterium 
of  chicken  cholera,  of  rabbit  septi- 
cemia and  the  bacillus  of  cholera 
suisepticus  so  closely  that  laboratory 
differentiations  are  extremely  difficult. 
The  bacterium  is  small,  with  rounded 
ends,   closely  resembling  a  diplococ- 


256 


BACTERIOLOGY. 


cus.  Involution  forms  may  appear. 
Shows  by  polar  staining,  decolorizes 
by  Gram,  produces  no  spores,  has  no 
flagella  and  is  nonmotile. 

THE    COl^ON-TYFHOID  DYSENTERY 
GBOUF  OF  BACII^I^I. 

The  organisms  belonging  to  this  group 
present  great  differences  in  their 
pathogenic  characters,  but  possess  so 
many  points  of  similarity  in  their 
morphological  and  biological  charac- 
teristics that  their  differentiation  be- 
comes extremely  difficult.  The  group 
considered  will  be  the  Coll  group,  the 
Enteritidis  group,  the  Dysentery 
group  and  the  Typhosus  group. 

The  differentiation  of  these  organisms 
is  important  in  that  certain  of  the 
bacilli  are  specifically  pathogenic, 
while  others  are  essentially  saphro- 
phytic  and  become  pathogenic  only 
under  exceptional  conditions. 

BACIUUS  COI^I  GBOUF. 

These  organisms,  sometimes  called 
"Lactose  Fermenters,"  are  frequently 
nonpathogenic,  for  man  but  may  be- 
,come  distinctly  pathogenic  under  cer- 
tain conditions.  Their  degree  of 
virulence  upon  inoculation  of  lower 
animals  varies  greatly. 

Motility  is  not  marked,  or  none.  Dex- 
trose and  lactose  are  fermented  with 
acid  production.  Milk  is  quickly 
coagulated  with  acid  production. 
Indol  is  not  produced  by  most  va- 
rieties. 

THE  BACIIiI^TJS  COZiI. 

Under  the  name  of  Colon  Bacilli  are 
grouped  a  number  of  varieties  which 
differ  from  one  another  in  minor 
characteristics,  but  correspond  in  cer- 
tain cardinal  points,  which  warrants 
their  consideration  under  one  heading. 

The  Bacillus  Coll  Commtmls  is  the  most 
prominent  type  of  the  group,  and 
therefore  will  be  chiefly  considered. 
This  organism  was  discovered  by 
Buchner  in  1885.  It  is  a  constant 
inhabitant  of  the  intestinal  canal  of 
human  beings  and  animals.  It  is 
occasionally  found  in  the  soil,  air, 
water  and  in  milk.  It  is,  in  fact, 
found  in  all  thickly  populated  neigh- 


BACTERIOLOGY.  257 

borhoods.  In  man,  the  bacillus  coll 
appears  normally  in  the  intestine, 
being  found  in  greatest  numbers  at 
or  about  the  ileocecal  valve  diminish- 
ing from  here  upward  to  the  duo- 
denum and  downward  as  far  as  the 
rectum.  The  organism  is  frequently 
found  in  the  tissues  and  in  blood  after 
death  without  visible  lesions  of  the 
intestinal  mucosa.  It  is  probable  that 
it  may  enter  the  circulation  a  few 
hours  before  death.  Extensive  inves- 
tigations have  been  carried  out  to  de- 
termine whether  or  not  the  presence 
of  the  organism  in  the  intestines 
possesses  a  definite  physiological 
function  of  advantage  to  its  post. 
This  question  has  not  been  definitely 
settled  though  it  might  be  stated  that 
the  function  of  the  organism  in  the 
intestine  is  not  inconsiderable  if  only 
because  of  its  possible  antagonism  to 
certain  putrefactive  bacteria,  as  has 
been  demonstrated  by  Bienstock.  The 
bacillus  coli  communis  is  a  short 
rod,  varying  in  thickness  from  one- 
third  to  one-fifth  its  length.  Under 
certain  conditions  of  cultivation,  it 
may  appear  more  slender  or  shorter 
and  even  coccoid  in  form.  It  usually 
appears  singly,  but  occasionally  in 
short  chains.  There  are  no  spores. 
When  first  isolated  from  the  body,  it 
may  be  extremely  motile,  while  old 
laboratory  strains  of  the  organism 
may  show  almost  no  motility.  Ordi- 
narily the  motility  of  the  colon 
bacillus  is  intermediate  between  these 
two  extremes.  Stains  readily  with 
the  ordinary  aniline  dyes  and  is 
decolorized  by  Gram. 
The  pathogenicity  of  the  organism  for 
animals  is  slight  and  varies  with 
the  different  strains.  If  1  cc.  of  a 
bouillon  culture  is  injected  intraperi- 
toneally  into  guinea  pigs,  death  will 
often  ensue.  If  large  doses  are  in- 
jected intravenously  into  rabbits, 
symptoms  of  violent  intoxication  are 
presented,  followed  by  death  in  from 
24  to  48  hours.  Moderate  doses 
inoculated  subcutaneously  usually 
produce  nothing  more  than  a  local 
abscess,  from  which  the  animal  re- 
covers. It  is  probable  that  death 
results  from  the  action  of  poisons  lib- 
erated  from   the   disintegrating  bac- 


258  BACTERIOLOGY. 


teria,  and  not  from  multiplication  of 
the  bacilli  themselves. 

In  man  a  largre  variety  of  lesions  pro- 
duced by  the  organisms  have  been 
described.  The  manner  in  which  the 
organism  becomes  pathogenic  is  not 
clear.  A  number  of  explanations  have 
been  advanced.  First,  that  whenever 
an  infection  is  produced  by  the 
bacillus  coli,  it  is  produced  by  one 
that  has  been  recently  acquired  from 
another  host;  second,  that  the  viru- 
lence of  the  organism  may  be  en- 
chanced  by  inflammatory  processes 
brought  about  by  other  organisms; 
third,  infection  may  possibly  take 
place  by  reason  of  a  reduction  in  the 
resistance  of  the  host.  Whatever  the 
cause  for  the  infection,  it  is  doubtful 
whether  septicemia  produced  by  the 
colon  bacillus  is  due  to  an  actual  pri- 
mary invasion  of  circulation  by  the 
bacillus,  although  a  few  unquestion- 
able cases  have  been  reported. 
Diseases,  such  as  cholera  nostras  and 
cholera  infantum,  have  been  attrib- 
uted to  these  organisms,  but  without 
being  supported  with  satisfactory  evi- 
dence. It  is  likely  that  in  most  of 
the  intestinal  diseases  formerly  attrib- 
.  uted  to  these  organisms  the  organism 
plays  but  a  secondary  part.  In  peri- 
tonitis following  perforation  in  the 
intestine,  the  organism  is  always 
present,  but  can  never  be  found  in 
pure  culture,  being  usually  accom- 
panied by  stpahylococci  and  strepto- 
cocci and  other  micro-organisms.  It 
is  therefore  hard  to  determine 
whether  or  not  these  bacilli  could  be 
considered  as  a  primary  cause  of  peri- 
tonitis. The  organism  may  give  rise 
to  a  mild  suppurative  process,  in  as 
much  as  it  is  able  to  proliferate 
within  the  peritoneum.  Welch  re- 
ports a  case  of  peritonitis  in  which 
the  bacillus  coli  was  isolated  in  pure 
culture.  The  organism  has  also  been 
isolated  from  liver  abscesses  from 
the  bile  and  from  the  center  of  gall 
stones,  consequently  inflammatory 
conditions  have  been  attributed  to  it 
in  these  situations. 

The  organism  is  found  more  frequently 
in  the  urine  than  any  other  organism. 
It  may  be  present  in  normal  indi- 
viduals.   It  is  frequently  seen  during 


BACTERIOLOGY.  259 


convalescence  from  typhoid.  It  may 
disappear  spontaneously  or  cystitis 
may  supervene  and  occasionally  an 
ascending  pyonephrosis.  The  organ- 
ism may  cause  localized  suppurations 
in  all  parts  of  the  body,  most  fre- 
quently seen,  however,  about  the  anus 
and  the  genitals. 

The  toxic  action  of  the  colon  bacillus 
is  due  to  endotoxins.  The  injection 
of  gradually  increased  doses  of  living 
or  dead  colon  bacilli  will  produce 
specific  bacteriolytic  agglutinating 
and  precipitating  substances.  The 
injection  of  any  specific  race  of  colon 
bacilli  produces  in  the  immunized  ani- 
mal high  agglutination  value  only  for 
the  individual  cuUure  used  for  im- 
munization. 

The  large  number  of  varieties  of  colon 
bacilli  described  during  the  early 
days  of  bacteriology  were,  in  many 
cases,  based  upon  a  temporary  de- 
pression of  one  or  another  cultural 
characteristic,  although  some  Were 
undoubtedly  closely  related.  They 
were,  however,  distinct  groups. 

The  constant  and  distinct  varieties  of 
the  bacillus  coli  do  not  occur.  The 
most  common  is  the  'bacillus  coli  com- 
munior  (Dunham),  and  is  believed  to 
be  more  abundant  in  the  human  and 
animal  intestines  than  the  coli  com- 
munis itself.  It  possesses  all  the 
cardinal  characteristics  of  the  colon 
group.  It  differs,  however,  from  the 
bacillus  coli  communis,  in  that  it 
produces  acid  from  the  saccahrose  as 
well  as  from  dextrose  and  lactose; 
whereas  the  coli  communis  does  not 
form  acid  or  gas  from  saccharose. 
The  Bacillus  Coli  Communis  is  an 
aerobe  and  facultative  anaerobe.  It 
will  grow  upon  all  of  the  ordinary 
media  at  a  temperature  ranging 
from  20°  to  40°  C,  optimum  37%**  C. 
In  broth,  general  clouding  with  later 
pellicle  and  a  light,  slimy  sediment  is 
formed.  Upon  agar,  gray  colonies 
appear  within  12  to  18  hours,  which 
gradually  become  more  and  more 
opaque.  The  surface  colonies  often 
show  a  characteristic  grape  leaf 
structure,  or  may  be  round  and  fiat 
and  show  a  definitely  raised,  glisten- 
ing surface.  Upon  agar  slant,  the 
growth  occurs  in  a  uniform  layer.  On 


260  BACTERIOLOGY. 


gelatin,  the  growth  is  rapid  with  no 
liquefaction.  On  potato,  the  growth 
is  abundant  and  of  a  gray  white, 
glistening  layer  which  later  turns  to 
a  yellowish  brown,  and  in  old  cultures 
often  to  a  dirty  greenish  brown. 
Indol  is  formed  in  peptone  solution. 
Milk  is  acidified  and  coagulated.  In 
lactose  litmus  agar,  acid  is  formed 
and  the  medium  becomes  red.  Gas 
bubbles  appear  along  the  line  of  the 
stab  inoculation.  Gas  is  formed  in 
dextrose,  lactose  and  mannite,  but  not 
in  saccharose.  Acid  and  gas  are 
formed  in  levulose,  lactose  and  mal- 
tose. 

The  isolation  of  the  colon  bacillus  from 
mixed  cultures''^  is  accomplished  by 
plating  upon  lactose  litmus  agar. 
Cultures  of  the  colon  bacillus  are 
characterized  by  an  odor  not  unlike 
that  of  diluted  feces.  The  acids 
formed  by  the  organism  from  sugar 
are  lactic,  acetic  and  formic  acids. 
The  gas  produced  is  chiefly  carbon 
dioxide  and  hydrogen. 

BACTERIUM    IkACTUS  AEBOGENES. 
(Bacillus  Aerog'enes). 

This  organism  was  first  isolated  by 
Escherich  (1885)  from  the  feces  of 
infants.  It  is  almost  constantly  pres- 
ent In  milk,  and  together  with  one  or 
two  other  microorganisms,  is  the 
chief  cause  of  the  ordinary  souring 
of  milk.  It  is  also  widely  distributed 
in  nature  in  feces,  in  water  and  in 
sewers.  It  is  anaerobe  and  facultative 
anaerobe.  Distinguished  from  the 
colon  bacillus  in  that  it  is  non-motile, 
very  seldom  forms  chains,  and  when 
cultivated  in  milk  it  possesses  a  dis- 
tinct capsule;  also  in  that  it  will  fer- 
ment polysaccharides  as  starch  and 
does  not  produce  indol. 

The  organism  is  but  slightly  pathogenic 
to  man.  It  is  almost  constantly  in 
the  human  intestines.  In  infants  it 
may  give  rise  to  flatulence  and  has 
been  known  to  produce  cystitis.  In 
rare  instances  it  has  formed  gas  in 
the  blood.  The  different  strains  of 
the  organism  vary  in  their  patho- 
genicity for  animals. 

It  grows  abundantly  at  a  temperature 
between  25"  to  30°  C  on  all  the  ordi- 


BACTERIOLOGY.  261 


nary  culture  media.  Upon  agar  and 
gelatin,  heavy,  white,  mucoid  colonies 
appear  which  have  a  tendency  to  con- 
fluence. In  broth,  there  is  general 
clouding  and  pellicle  formations;  a 
sour  or  cheesy  odor.  Upon  potato, 
growth  is  heavy  and  gas  is  formed. 
Milk,  coagulated  and  acidified.  The 
organism's  chief  characteristic  is  that 
it  is.  capable  of  producing  a  large 
amount  of  acid,  chiefly  lactic,  and  of 
being  able  to  withstand  this  quan- 
tity of  acid  without  being  in- 
jured. All  carbohydrates,  except  sac- 
charose, are  fermented  with  the 
formation  of  gas. 

BACII^XiUS    MUCOSUS  CAFSUl^ATXTS. 
(Friedlauder's  Bacillus.  Bacterium 
Pneumonia,  Fneumobacillus  ) . 

Friedlander's  Bacillus,  the  Bacillus  of 
Rhinoscleroma,  the  Bacillus  of  Ozena, 
etc.,  together  with  a  number  of  bac- 
teria reported  as  allied  to  Friedland- 
er's, mainly  upon  morphological 
grounds,  are  classified  together  as  the 
"Friedlander  group"  of  organisms. 

This  organism  was  discovered  by  Fried- 
lander  in  1882,  and  believed  by  him  to 
be  the  cause  of  lobar  pneumonia. 
He  described  it  as  a  micrococcus. 
Researches  by  Frankel  and  Weichsel- 
baum  proved  this  to  be  a  short,  in- 
capsulated  bacillus,  which  occurred 
in  lobar  pneumonia  on  rare  occa- 
sions only.  The  organism  is  a  short, 
plump  bacillus  with  rounded  ends, 
varying  greatly  in  size,  even  in  the 
same  culture.  In  animal  and  human 
lesions  the  organism  is  almost  coccoid 
in  form.  It  appears  singly  in  diplo- 
form  or  in  short  chains.  They  are 
nonmotile.  It  is  surrounded  by  a  cap- 
sule in  animals  when  taken  from  ani- 
mal fluid  and  sometimes  in  the  smears 
from  agar  or  gelatin.  The  capsule  is 
two  or  three  times  the  size  of  the 
bacillus,  and  when  seen  in  chains  or 
in  groups,  several  bacilli  may  be  en- 
closed within  one  capsule.  The  cap- 
sule will  disappear  in  prolonged  cul- 
tivation on  agar  or  gelatin.  It  stains 
easily  with  the  ordinary  stains,  but  is 
decolorized  by  Gram. 

It  causes  pneumonia  probably  in  about 
7  or  8  per  cent,  of  all  cases  in  this 


262  BACTERIOLOGY. 


country.  When  causing  the  disease, 
the  pneumonia  is  extremely  severe 
and  usually  fatal.  It  has  been  found 
in  ulcerative  stomatitis  and  nasal 
catarrh.  It  has  been  reported  as  oc- 
curring- in  severe  tonsilitis  in  children, 
in  the  pus  from  suppurations  in  the 
antrum  of  Highmore  and  nasal 
sinuses  and  in  cases  of  ozena,  believed 
by  Abel  to  be  the  specific  cause.  It 
has  been  found  in  empyemic  fluid,  in 
pericardial  exudate  and  in  spinal  fluid. 
Cases  of  septicemia  have  been  de- 
scribed, caused  by  Friedlander's 
bacillus.  It  has  been  believed  to  be 
associated  with  some  forms  of 
diarrheal  enteritis  in  that  it  is  an 
occasional  inhabitant  of  the  normal 
intestine. 

It  is  pathogenic  for  mice  and  guinea 
pigs,  also  for  rabbits.  Inoculated  into 
susceptible  animals,  it  will  produce 
inflammation  and  death  by  septicemia. 
Intraperitoneal  inoculatipn,  a  mucoid 
stringy  exudate  is  found  which  is 
characteristic. 

Immunization  with  graded  doses  of 
dead  bacillus  has  been  produced  in 
isolated  cases.  Specific  agglutinins 
,in  the  immune  serum  have  occasion- 
ally been  found,  potent  only  against 
the  particular  strains  used.  The 
organism  is  an  aerobe  and  facultative 
anaerobe  and  grows  easily  on  all  cul- 
ture media.  Temperature  ranges 
from  18°  to  40°  C,  optimum  37  V^''  C. 
On  agar,  grayish  white  mucous  light 
colonies  of  a  slimy,  semifluid  consis- 
tency appear.  After  three  or  four 
days  a  tendency  to  confluence  causes 
a  large  part  of  the  surface  to  be  cov- 
ered with  a  film  of  glistening,  sticky 
exudate,  which,  if  fished,  comes  ofC 
in  a  tenacious,  stringy  matter.  In 
broth,  there  is  rapid  abundant  growth 
with  pellicle  formation.  General 
clouding  and  later  a  stringy  sediment. 
Stab  cultures  in  gelatin  show  at  first 
a  white  line  of  growth  along  the 
course  of  the  puncture;  later,  there 
appears  a  grayish  glucose  droplet  on 
the  surface,  which  enlarges  and  gives 
the  growth  a  nail-like  appearance, 
which  is  regarded  as  diagnostic.  The 
gelatin  is  not  liquefied.  On  potato, 
an  abundant  growth  appears  of  a 
slightly    brown   color.     There   is  no 


BACTERIOLOGY.  263 

indol  formed  in  peptone  solution.  In 
milk,  the  growth  is  rapid,  with  irreg- 
ular coagulation.  All  carbohydrates 
(except  lactose)  are  fermented  with 
formation  of  gas. 

BACIZil^US  OF  HHINOSCIiEBOMA. 

The  bacillus  was  discovered  by  •  von 
Prisch  in  1882.  It  produces  a  disease 
called  in  rhinoscleroma  in  man,  which 
consists  of  a  slowly  grown  granu- 
lomatous inflammation,  located  at  the 
external  larynx,  or  the  mucosa  of 
nose,  mouth,  pharynx  or  larynx. 
Within  the  interior  of  the  lesions  are 
many  large,  swollen  cells,  within 
which  the  bacilli  lie;  also  in  the  in- 
tracellular spaces.  The  disease  is 
rare  in  America.  It  is  slowly  pro- 
gressive and  comparatively  intract- 
able to  surgical  treatment,  seldom 
affecting  the  general  health  unless  by 
obstruction  to  the  air  passages.  The 
bacillus  may  be  isolated  from  the 
lesions.  Morphologically  and  cultur- 
ally, it  is  almost  identical  with  Fried- 
lander's  bacillus.  It  differs  from 
Priedlander's  bacillus  in  forming  no 
gas  in  dextrose  bouillon,  and  pro- 
ducing no  acid  in  lactose  bouillon  and 
never  coagulating  milk. 

BACIIkLnS  OZENA. 

Abel  and  others  have  shown  that  an 
organism  morphologically  and  cultur- 
ally almost  identical  with  the  bacillus 
mucosus  capsulatus  is  nearly  always 
present  in  ozena.  While  he  states 
that  it  does  not  form  gas  in  dextrose 
bouillon  and  is  less  pathogenic  for 
mice  than  is  the  bacillus  of  Fried- 
lander,  it  cannot  be  definitely  stated 
as  to  whether  it  is  a  separate  species 
or  merely  an  atypical  form  of  the 
bacillus  of  Friedlander. 

BACII^IiUS  ENTERITIDIS  GROUP. 

Most  members  of  this  group  of  organ- 
isms are  under  certain  conditions  dis- 
tinctly pathogenic  for  many  of  the 
lower  animals  and  for  man.  The 
motility  is  usually  marked.  Dextrose 
is  fermented  with  gas  formation. 
Lactose  is  not  fermented.  Milk  is  not 
coagulated.  No  indol  or  only  a  slight 
amount  is  produced  (bacillus  alka- 
ligenese  does  not  ferment  sugars). 


264  BACTERIOLOGY. 

Gartner's  discovery  in  1888  of  the 
bacillus  enteritidis  in  association 
with  epidemics  of  meat  poisoning 
gave  impetus  to  the  study  of  a  num- 
ber of  bacteria  resembling  in  many 
characteristics  the  colon  or  typhoid 
bacilli. 

They  are  often  spoken  of  as  "group  of 
intermediates"  and  classified  as  inter- 
mediate between  the  colon  and  the 
typhoid  types. 

By  reason  of  the  pathological  conditions 
with  which  they  have  been  associated, 
the  terms  "hog-cholera  group,"  "en- 
teritidis group,"  "paracolon  group" 
or  "paratyphoid  group,"  were  applied 
to  the  chief  members  under  investi- 
gation. 

The  microorganisms  are  morpholog- 
ically indistinguishable  from  the 
colon  and  typhoid  bacilli.  Patho- 
genically  they  have  attracted  atten- 
tion in  their  connection  with  meat 
poisoning  and  with  protracted  fevers 
that  are  indistinguishable  from  mild 
typhoidal  infections. 

Bacillus  Enteritidis  (Gartner)  was  dis- 
covered and  isolated  from  the  meat 
of  a  cow,  the  ingestion  of  which  had 
•  produced  the  symptoms  of  acute 
gastrointestinal  catarrh  in  fifty-seven 
people.  The  organism  was  demon- 
strated in  the  spleen  and  in  the  blood 
of  one  patient  who  died  from  the  dis- 
ease. The  organism  is  actively  mo- 
tile, forms  no  indol  and  produces  gas 
in  dextrose  media.  If  fed  to  mice, 
guinea  pigs,  rabbits  and  sheep,  it  will 
induce  acute  gastrointestinal  symp- 
toms. The  bodies  of  the  organisms 
contain  an  extremely  toxic  substance 
which  differs  from  the  endotoxins  of 
other  bacteria  in  that  it  is  extremely 
resistant  to  heat.  Sterilized  cultures 
have  the  same  pathogenic  effect  as 
the  living  cultures. 

Bacillus  Morselle,  described  by  Van 
Ermengem  in  1891  in  an  epidemic  of 
meat  poisoning  at  Morselle,  differing 
slightly  in  minor  characteristics,  is 
almost  identical  with  Gartner's  bacil- 
lus. 

Bacillus  Psittacosis,  isolated  by  Nocard 
in  1892  from  infections  in  parrots, 
showed  a  close  resemblance  to  Gart- 
ner's bacillus. 


«  BACTERIOLOGY. 


265 


Bacillus  Typhimurium,  isolated  by 
Loeffler,  was  in  1893  shown  to  be  sim- 
ilar to  Gartner's  bacillus  and  also  to 
the  so-called  "hog-cholera  bacillus," 
by  T.  Smith  and  Moore  from  their 
studies  of  the  disease  of  swine.  They 
first  used  the  term  of  "hog  cholera" 
group. 

Paracolon  and  Paratyphoid  Group  were 
introduced  by  Gilbert  in  1893  to  des- 
ignate the  organisms  of  this  group 
resembling  more  nearly  the  biolog- 
ical characters,  the  colon  bacillus  on 
the  one  hand  and  typhoid  bacillus  on 
the  other. 

Bacillus  bovls  morbificans  was  isolated 
by  Basenan  in  1894  in  an  epidemic 
of  meat  poisoning;  differing  slightly 
in  minor  characteristics,  it  is  almost 
identical  with  Gartner's  bacillus. 

Paracolon  Bacillus  was  isolated  by 
Widal  and  Nobecourt  in  1897  from 
an  esophageal  abscess  following 
typhoid  fever.  This  organism  showed 
a  close  resemblance  to  Gartner's 
bacillus,  and  following  the  Gilberts' 
suggestion  they  named  it  "B.  para- 
colon." 

Gwyn,  in  1898,  isolated  an  organism 
from  the  blood  of  a  patient  who  pre- 
sented all  the  symptoms  of  typhoid 
fever,  but  the  patient's  serum  did  not 
have  any  agglutinating  power  for  the 
bacillus  typhosus.  Its  culture  char- 
acteristics were  similar  to  those  of 
Gartner's  bacillus  which  was  agglu- 
tinated by  the  patient's  serum.  He 
called  it  "paracolon  bacillus."  The 
paracolon  and  paratyphoid  can  be  dis- 
tinguished without  difficulty  from  the 
typhoid  bacillus.  They  produce  gas 
in  gl\icose  media,  and  in  this  respect 
they  differ  from  typhoid,  but,  unlike 
B.  coli,  they  do  not  produce  gas  from 
lactose,  coagulated  milk,  or,  as  a  rule, 
from  indol. 

Agglutination  tests  applied  to  the  in- 
termediates show  that  the  members 
of  the  paracolon  group  do  not  all 
show  mutual  reactions,  and  the 
group,  like  the  B.  coli,  is  therefore 
composed  of  a  number  of  distinct 
races.  The  paratyphoids,  most  of 
which  have  been  isolated  from  cases 
simulating  typhoid  fever,  belong 
chiefly  to  two  strains.  An  active 
serum  prepared  from  either  strain  of 


266  BACTERIOLOGY. 


the  bacilli  will  agglutinate  all  the 
others  of  that  strain.  They  are  des- 
ignated as  type  A  and  type  B. 

A  similar  organism  was  isolated  by 
Gushing,  in  1900,  from  a  costo- 
chondral  abscess  during  convalescence 
from  typhoid  fever. 

En,cillus  Icteroides,  associated  by  Sana- 
relli  with  yellow  fever,  was  shown  by 
Reed  and  VarroU,  in  1899,  to  be  cul- 
turally similar  to  the  bacillus  of  hog- 
cholera. 

Bacillus  Paratyphoid,  Schottmuller,  in 
1900,  isolated  bacilli  from  five  cases 
which  corresponded  to  bacilli  pre- 
viously described. 

Cultural  and  agglutination  studies  of 
the  organisms  obtained  showed  that 
they  could  be  divided  into  two  sim- 
ilar yet  distinctly  different  types;  one 
of  them  very  close  to  the  typhoid 
type  (B.  Paratyphoid);  the  other, 
closer  to  the  Gartner  bacillus. 

Type  A  has  been  isolated  from  the  nor- 
mal intestines  of  animals  by  Morgan 
and  is  not  considered  very  important 
as  a  causative  agent  of  human  dis- 
ease. Kutscher  therefore  suggests 
that,  except  in  rare  instances,  this 
•organism  is  a  nonsaphrophtic  para- 
site. Type  B  not  infrequently  pro- 
duces an  infection. 

Clinically,  the  diseases  caused  by  this 
class  of  bacteria  may  be  divided  into: 

Group  1.  Those  which  fall  into  the 
category  of  meat  poisoning  (Para- 
colon) more  like  those  due  to  B. 
Gartner,  having  sudden,  violent  onset 
of  gastrointestinal  symptoms  directly 
following  the  ingestion  of  meat,  and 
characterized  by  profound  toxemia. 

Group  2.  Those  in  which  the  disease 
simulates  a  mild  form  of  typhoid 
fever,  lasting  from  twelve  to 
eighteen  days,  and  differing  only 
by  the  absence  of  the  specific  agglu- 
tination reaction  for  typhoid  bacilli. 

Bacillus  Alkalig'enes.  This  bacillus  re- 
sembles somewhat  a  colon  bacillus 
which  has  lost  its  power  to  ferment 
sugars.  Morphologically  and  cultur- 
ally it,  is  more  like  the  typhoid 
bacillus.  It  ferments  no  sugars.  It 
is  frequently  present  in  the  intestines 
and  may  have  pathogenic  properties. 

Bacillus  Cliolerae  Suis  (Bacillus  of  Hogr- 
Cholera).     This  organism  is  an  ac- 


BACTERIOLOGY.  267 

tively  motile  bacillus;  grows  readily 
in  bouillon;  renders  milk  at  first 
slightly  acid,  then  strongly  alkaline; 
dissolves  casein  and  ferments  dex- 
trose with  acid  production.  It  is 
found  almost  regularly  present  in 
hogs,  sick  of  cholera,  but  is  not  the 
essential  cause  of  the  disease.  Al- 
though it  is  not  an  essential  factor 
in  exciting  hog-cholera,  it  is  believed 
to  be  of  importance  as  an  added  in- 
fection. It  is  pathogenic  for  hogs, 
causing,  when  fed,  a  fatal  enteritis. 
Theobald  Smith  and  Moore,  in  1893, 
studied  this  disease  and  noted  a  great 
similarity  between  the  organism,  the 
bacillus  of  the  Gartner  group  and  the 
bacillus  typhi  murium  isolated  by 
Loeffler. 

Bacillus  of  Swine  Plague.  This  is  a 
nonmotile  bacillus  which  grows 
feebly  in  bouillon.  Does  not  coagu- 
late milk  and  ferments  glucose  with- 
out production  of  gas.  When  fed  to 
pigs  it  does  not  usually  cause  illness. 

It  is  closely  related  to  the  hemorrhagic 
septicemic  group. 

THE  BACUZiUS  DYSENTERY 
GROUP. 

This  group  of  organisms,  often 
grouped  with  bacillus  typhosus  are 
pathogenic  for  man  and  by  inocula- 
tion less  pathogenic  for  animals. 
The  organisms  are  nonmotile,  fer- 
ment dextrose  without  formation  of 
gas,  do  not  ferment  lactose,  do  not 
coagulate  milk  nor  produce  indol. 
The  bacillus  dysenteria  (Shiga)  does 
not  ferment  mannite.  The  bacillus 
paradysenteria  (Park)  ferments  mal- 
tose and  mannite.  The  paradysen- 
teria (Plexner)  ferments  mannite 
only. 

Bacillus  Dysenteria  (Shigra).  The  etiol- 
ogy of  dysentery  was  obscure  until 
Shiga  (1898)  found  a  bacillus  in  the 
stools  of  patients  suffering  with 
dysentery  which  had  not  before  been 
identified.  It  was  present  in  all 
the  cases  of  epidemic  dysentery  ex- 
amined but  was  not  found  in  the  stools 
of  healthy  persons. 

The  blood  of  dysenteric  patients  agglu- 
tinated the  bacilli  which  were  iso- 
lated, but  the  organisms  were  not 
agglutinated »  to  any  such  degree  by 


268  BACTERIOLOGY. 


serum  from  healthy  individuals.  The 
org-anism  is  a  short  rod  similar  to  the 
colon  group  of  org^anisms.  Stains  easily 
with  the  aniline  dyes  with  the  ends 
showing  a  tendency  to  deeper  stain- 
ing than  the  center. 

It  decolorizes  by  Gram's.  No  spores 
or  flagella  have  been  demonstrated. 
On  gelatin  the  colonies  appear  more 
like  the  typhoid  than  the  colon 
bacilli.  It  is  not  liquefied.  On  agar 
the  colonies  resemble  those  of  the 
typhoid  bacilli. 

On  potato,  a  delicate,  scarcely  visible, 
brownish  growth  is  formed.  In  bouil- 
lon, a  diffuse  cloudiness  is  formed 
with  a  slight  deposit  appearing 
after  some  days.  Occasionally  a 
pellicle  is  formed. 

Litmus  Milk  becomes  a  pale  lilac  after 
24  hours,  which  returns  to  the  orig- 
inal color  after  three  to  eight  days. 
Neutral  red  agar  is  not  changed. 

It  does  not  form  indol,  except,  perhaps, 
in  intestine,  or  ferment  mannite,  mal- 
tose or  saccharose.  Animals  injected 
with  this  type  of  org-anism  produce 
specific  ag-glutinins  which  only  in  a 
small  way  combine '  with  the  other 
type  of  the  group. 

In  man,  the  organisms  produce  an  acute 
dysentery  with  symptoms  of  cramps, 
diarrhoea  and  tenesmus.  The  stools, 
at  first  feculent,  then  seromucous, 
become  bloody  or  composed  of  coffee- 
ground  sediment.  At  the  height  of 
the  disease  there  are  ten  to  fifty 
stools  in  the  twenty-four  hours.  The 
blood  usually  disappears  after  from 
two  to  seven  days.  The  disease  is 
especially  limited  to  the  mucous  mem- 
brane of  the  large  intestines.  The 
vessels  of  the  surfaces  appear  con- 
gested and  prominent.  The  mucous 
membrane  is  covered  with  a  yellowish 
mucous  and  seems  to  be  absent  in 
places.  The  solitary  follicles  are  en- 
larged, especially  in  the  sigmoid 
flexure,  and  in  some  instances  are 
depressed  and  appear  to  be  necrotic 
in  their  center.  Microscopically,  the 
mucous  glands  are  normal  except 
over  the  solitary  follicles,  where  they 
are  slightly  broken  down  and  contain 
polynuclear  leukocytes.  The  capil- 
laries of  the  follicles  are  extremely 
congested.     The  submiicosa  is  thick- 


BACTERIOLOGY.  269 

ened  and  slightly  edematous.  The 
connective  tissue  cells  have  under- 
gone a  slight  hyaline  degeneration. 
The  deeper  coats  of  the  intestine  are 
not  involved. 

The  small  intestine  seems  to  be  slightly 
distended.  The  mesenteric  glands  are 
large  and  red.  Peyer's  patches  are 
swollen  slightly,  but  without  ulcera- 
tion. Microscopically,  the  mucous 
membrane  appears  normal. 

In  the  severe  cases  the  entire  lumen 
of  the  intestines  may  be  filled  with  a 
pseudomembrane  of  a  diphtheretic 
character.  In  young  children  the 
lesions  appear  to  be  more  superficial 
even  in  fatal  cases. 

Animals  injected  intravenously  with 
the  organism  show  symptoms  of 
diarrhoea  and  paralysis,  which  is  fol- 
lowed by  death.  Animals  are  likewise 
very  sensitive  to  killed  cultures. 
Autopsies  on  animals  killed  from  in- 
jections into  the  peritoneum  of  living 
or  dead  bacilli  show  a  hyperemic  peri- 
toneum, the  cavity  of  which  is  more 
or  less  filled  with  serous  or  bloody 
serous  exudate.  The  liver  may  be  cov- 
ered with  a  fibrinous  mass,  the  small 
intestine  filled  with  fluid,  the  large 
intestine  usually  empty,  and  the 
mucous  membrane  of  both  hyperemic 
and  may  sometimes  be  hemorrhagic. 
Subcutaneous  injections  of  the  dead 
or  living  organisms  produces  infiltra- 
tions of  tissue  and  frequently  abscess 
formation.  The  organism  produces 
both  an  extracellular  and  a  cellular 
toxin. 

Bacillus  Paradysentery   (Farke).  "A." 

In  1902,  Parke  and  Dunham  described 
an  orgariism  which  they  found  in  a 
small  epidemic  of  dysentery  occuring 
in  men  which  differed  somewhat  from 
the  organisms  previously  described, 
in  that  it  produces  endol.  The  organ- 
isms ferment  mannite  with  the  pro- 
duction of  acid,  but  does  not  act 
upon  maltose  or  saccharose.  Animals 
injected  with  this  organism  develop 
immune  bodies  and  agglutinins  that 
are  specific  for  this  type. 
Bacillus  Paradysentery  (Plexner).  "B." 
Flexner  in  1899,  while  investigating 
the  dysentery  in  the  Philippines,  iso- 
lated an  organism  which  corresponds 
to  Shiga's  organism,  but  differs  from 
the  other  in  that  it  produces  endol. 


270  BACTERIOLOGY. 


ferments  mannite  and  acts  strongly 
upon  maltose  and  feebly  upon  saccha- 
rose. This  type  of  organism  is  near- 
est to  the  Colon  group. 

The  two  mannite  fermenting  types  are 
widely  scattered  over  the  world  and 
caused  epidemics  of  dysentery  of  a 
milder  type  than  that  produced  by 
the  Shiga  organism  and  have  been 
described  by  many  investigators. 
These  two  types  have  also  been 
described  at  times  in  mixed  infec- 
tions where  dysentery  symptoms  are 
almost  or  entirely  absent. 

Passive  immunization  of  animals  and 
human  beings  with  the  serum  of  im- 
munized horses  has  been  attempted 
by  Shiga,  Kruse  and  others,  who  have 
reported  a  reduction  of  mortality  by 
the  use  of  such  sera.  Todd  has  dem- 
onstrated the  neutralization  of  the 
solutions  of  toxin  by  an  immune 
serum. 

By  reason  of  the  different  varieties  of 
dysentery  bacilli,  polyvalent  sera  is 
of  considerable  value.  It  is  given 
subcutaneously  in  20  cc.  doses  once 
or  twice  a  day  for  several  days,  or 
until  convalescence  is  established. 

BACUI^US  TYPHOSUS  GROUP. 

The  Bacillus  Typhosus  is  an  actively 
motile  organism  pathogenic  for  man 
and  less  pathogenic  by  inoculation  for 
lower  animals.  Dextrose  is  fermented 
without  gas  formation;  lactose  not 
fermented;  milk  not  coagulated  and 
no  formation  of  indol. 

The  organism  was  discovered  by  Ebert 
(1880)  in  the  spleen  apd  diseased 
areas  of  the  intestine  of  typhoid 
cadavers. 

It  was  obtained  in  pure  culture  by 
Gaffky  in  1884. 

The  organism  is  a  short  plump  rod 
having  rounded  ends.  Under  favor- 
able conditions  it  is  actively  motile. 
The  degree  of  motility  varies  in  dif- 
ferent cultures.  The  flagella  are 
preipherally  arranged  of  twelve  or 
more  in  number.  Many  of  the  shorter 
forms  have  but  a  single  terminal 
flagellum. 

The  bacillus  stains  a  little  less  in- 
tensely with  the  ordinary  aniline  dyes 
than  do  most  other  bacteria.  Bipolar 


BACTERIOLOGY.  271  * 

staining    is    sometimes    marked  and 
decolorized  by  Gram's. 

The  organism  is  aerobic  and  facultative 
anaerobic  bacillus  developing  best  at 
37°  C.  Its  growth  is  retarded  above 
40°  C  and  below  30°  C.  Below  10°  C 
its  growth  almost  ceases.  It  does  not 
form  spores.  Some  of  the  bacilli  are 
killed  within  a  few  hours  when  dried, 
though  a  few  will  remain  alive  for 
months  under  the  same  conditions. 
Usually  they  are  killed  by  an  ex- 
posure to  60°  C  for  one  minute. 

In  natural  water  it  remain^  alive  for 
36  days  (Klein). 

In  ice  it  may  remain  alive  for  three 
months  (Prudden). 

It  is  killed  by  1-500  bichloride  or  5% 
carbonic  acid  within  five  minutes. 

Upon  agar  plates,  small  grayish  col- 
onies appear  within  18  to  24  hours. 

These  colonies  are  first  transparent, 
later  they  become  opaque.  Upon 
agar  slants,  the  transparent,  filiform 
grayish  streak  is  formed. 

Upon  gelatin  plates,  characteristic  irreg- 
ular (grape-leaf)  transparent,  bluish- 
white  colonies  appear.  Magnified,  they 
are  of  homogenous  structure  marked 
by  a  delicate  network  of  furrows. 

As  the  colonies  grow  older  they  grow 
heavier,  become  more  opaque  and  lose 
their  early  differential  value. 

In  gelatin  stab  cultures  the  growth  is 
mostly  on  the  surface  as  a  thin  scal- 
loped extension  which  gradually 
reaches  the  sides  of  the  tube.  In  the 
stab  proper  there  is  but  a  limited 
growth  of  yellowish  brown  color. 
Gelatin  is  not  liquefied. 

Bouillon  is  uniformly  clouded.  When 
the  medium  is  slightly  filkaline  a 
delicate  pellicle  may  be  formed  after 
18  to  24  hours'  growth. 
.  On  potato,  a  characteristic  almost  in- 
visible growth  appears  after  24  to  48 
hours  which  usually  covers  the  sur- 
face of  the  medium,  though  it  may  be 
restricted  to  the  point  of  inoculation. 
The  growth  may  also,  however,  be 
quite  heavy  and  of  a  yellowish-brown 
color  with  a  greenish  halo  like  that 
of  B.  Coli.  Milk  is  not  coagulated. 
The  neutral  violet  color  In  litmus 
whey  becomes  more  red  during  the 
first  48  hours;  the  fluid  remaining 
clear. 


272  BACTERIOLOGY. 


In  Dunham's  peptome  solution  there  is 
no  production  of  indol. 

The  organism  does  not  produce  gas 
when  grown  in  dextrose,  mannite,  lac- 
tose and  saccharose  Droth,  but  it  does 
produce  acid  in  dextrose,  levulose,  gel- 
actose,  mannite,  maltose  and  dextrin 
broth. 

In  shake  or  stab  cultures  of  Rothberg- 
er's  neutral  red  no  change  is  pro- 
duced while  the  colon  group  reduce 
the  red,  decolorize  the  media  and  pro- 
duce gas. 

When  inje\3ted  into  animals,  no  typicai 
pathological  changes  are  '  produced. 
The  sickness  or  fatal  results  after 
such  injections  can  be  attributed  to 
the  toxemia  produced  by  the  endo- 
toxins liberated  from  the  dead  bac- 
teria. 

Animals  inoculated  subcutaneously  by 
bacilli,  freshly  obtained  from  typhoid 
cases,  rapidly  die. 

In  the  peritoneal  cavity  they  may  in- 
crease with  the  production  of  a  fatal, 
peritonitis.  If  the  bacilli  are  accus- 
tomed to  the  animal  body  the  viru- 
lence may  be  so  increased  as  to  prove 
fatal  to  the  animal  when  injected 
.with  very  small  cultures. 

The  organism  produces  in  man  an  in- 
fectious disease  in  which  the  organ- 
isms pass  into  the  blood,  and  by  this 
channel  they  pass  to  all  parts  of  the 
body  and  become  localized  in  the  tis- 
sue such  as  the  bone  marrow,  lym- 
phatic tissues  and  spleen,  liver  and 
kidneys. 

The  lesions  of  the  intestine  consist  of 
an  inflammatory  enlargement  of  the 
solitaryjs  and  agminated  lymph 
nodules.  In  the  more  severe  cases 
the  hyperplasia  is  frequently  followed 
by  ulceration  and  necrosis. 

Ulceration  and  sloughing  may  involve 
the  muscular  and  peritoneal  coats 
with  the  production  of  perforation. 
Peritonitis  and  death  usually  follows, 
though  in  rare  instances  adhesions 
may  close  the  perforation. 

The  mesenteric  lymph  nodes  undergo 
changes  like  those  of  the  ilium.  The 
spleen  is  enlarged  by  reason  of  con- 
gestion and  hyperplasia.  The  liver 
and  to  less  extent  the  kidney  are  apt 
to  show  foci  of  cell  proliferation. 


BACTERIOLOGY.  273 

The  typhoid  bacillus  may  in  rare  cases 
act  as  "pus  producer." 

The  complications  occuring  in  typhoid 
fever  are  usually  due  to  secondary 
or  mixed  infections  with  the  sta- 
phylococcus, pneumococcus,  strepto- 
coccus, pyocyanens  and  colon  bacillus. 

The  organism  is  present  in  the  urine 
of  typhoids  in  about  20%  of  cases 
during  the  third  or  fourth  week. 
When  pneumonia  is  caused  by  B. 
typhosus  it  can  be  found  in  the  spu- 
tum. 

During  typhoid  fever  the  organism  is 
always  found  in  the  gall  bladder.  The 
organism  usually  disappears  from  the 
body  in  the  fourth  or  fifth  Week,  but 
may  remain  for  months  or  years  in 
the  urine  and  throughout  life  in  the 
gall  bladder. 

Abscesses  have  been  found  one  year 
after  recovery  from  typhoid  fever. 

One  to  five  per  cent,  of  individuals  hav- 
ing had  typhoid  continue  to  pass 
typhoid  bacilli  for  years,  maybe  for 
life. 

A  number  of  so-called  typhoid  carriers 
have  been  reported  which  if  not  de- 
tected are  very  dangerous  *  as  con- 
stant spreaders  of  typhoid  fever.  The 
treatment  of  these  cases  has  not  been 
satisfactory.  Medicinal  treatment 
and  immunization  have  yielded  slight 
results. 

Recovery  from  typhoid  fever  produces 
an  immunity  which  may  last  for 
years,  except  in  about  2%  of  cases. 
The  second  attack  in  these,  however, 
is  usually  of  a  mild  type. 

Serum  of  animals  immunized  possesses 
bactericidal  and  feeble  antitoxic  prop- 
erties against  B.  typhosus,  and  an 
attempt  has  been  made  to  treat 
typhoid  by  this  method,  and  although 
good  results  have  been  reported  by  a 
number  of  men  the  majority  have 
found  little  or  no  value  in  its  use. 

Where  danger  of  typhoid  infection 
exists,  the  use  of  protective  vaccines 
advocated  by  Wright  are  indicated. 
(See  typhoid  vaccine.) 

Vaccination  durin-g  the  course  of  the 
fever  has  been  advised  by  certain 
individuals,  but  the  results  obtained 
by  Richardson  does  not  show  any 
effect  except  that  relapses  seem  to 
be  less. 


274  BACTERIOLOGY. 

For  the  diagnosis  of  typhoid  see  the 
Gruber-Widal   reaction   (Widal  test). 

DISEASES    DUE    TO    THE  HIGHER 
BACTERIA. 

^eptothrlz.  Forms  which  appear  as 
simple  threads  without  branching. 
Members  of  this  group  have  been 
found  associated  with  certain  inflam- 
mation of  the  mouth  and  pharynx. 
The  organisms  were  identified  by 
morphology. 

None  of  the  inflammations  were  accom- 
panied by  severe  systemic  symptoms 
and  the  organisms  may  be  regarded 
as  comparatively  harmless  sapro- 
phyteg  appearing  in  connection  with 
some  other  specific  inflammation. 

Cladothrix.  Thread-like,  forms  with 
false  branching,  due  to  the  fragmen- 
tation of  the  threads. 

By  reason  of  the  difficulty  in  difCeren- 
tiation  of  this  form  from  the  strepto- 
thrix,  it  is  likely  that  most  cases  of 
infection  ascribed  to  these  organisms 
have  really  been  due  to  streptothrix 
infection.  *A  rigid  difCerention  of 
true  and  false  branching  only  can 
determine  whether  or  not  cladothrix 
infection  may  occur. 

Streptothrix.  (Nocardia.)  Forms  with 
numerous  true  branches  and  spores 
which  usually  appear  in  chains. 

Numerous  cases  of  infection  of  various 
parts  of  the  body  of  man  and  animal 
have  been  reported.  A  member  of 
this  group  has  been  described  by 
Nocard  as  the  etiological  factor  in.  a 
disease  "farcies  du  boeuf,"  occuring 
among  cattle  in  Guadeloupe. 

Trevisan  proposed  the  name  "Nocardia" 
for  this  organism,  and  Wright  calls 
attention  to  the  misuses  of  the  term 
"streptothrix"  and  points  out  +hat  the 
term  Nocardia  should  be  used  in  its 
place. 

Members  of  this  group  have  also  been 
reported  in  the  pus  from  a  cerebral 
abscess,in  pulmonary  disease  simu- 
lating tuberculosis,  suppuration  of 
bone  and  of  the  skin  and  the  intes- 
tinal canal.  Streptothrices  vary  con- 
siderably in  morphology.  In  infec- 
tious lesions  they  most  aften  appear 
as  rods  and  filaments  with  branches. 
Sometimes  the  filaments  may  be  long 
and    intertwined,    and    branch  may 


BACTERIOLOGY.  275 

show  club-shaped  ends.  Young"  cul- 
tures may  consist  of  rod-shaped  forms 
not  unlike  bacilli  of  the  diphtheria 
group. 

They  stain  easily  with  Loeffler's  methy- 
lene blue  or  aqueous  fuchsin.  Culti- 
vation upon  agar  and  gelatin  plates 
has  been  made.  Grayish-white,  glis- 
tening, flat  colonies  appear  at  the  end 
of  four  or  five  days.  In  bouillon,  a 
floculent  precipitate  and  surface  pel- 
licle is  formed  of  the  thread,  without 
clouding. 

The  organisms  will  grow  readily  upon 
fresh,  sterile  kidney-tissue  of  rabbits. 
When  cultures  are  inoculated  into 
rabbits  and  guinea  pigs,  subcutaneous 
abscesses,  bronchopneumonia,  and 
suppuration,  according  to  the  mode 
of  infection  may  be  produced. 

Actinomyces  (Ray  fungus)  is  charac- 
terized by  the  formation  of  club- 
shaped  ends  and  the  stellate  arrange- 
ment of  its  threads. 

The  organism  was  first  observed  by 
Bollinger  (1877)  in  diseased  cattle 
and  by  Israel  (1878)  in  man. 

The  organisms  appear  in  the  pus  from 
discharging  lesions  as  small  granular 
bodies,  resembling  sulphur  granules, 
visible  microscopically. 

They  are  ordinarily  soft  and  can  be 
easily  crushed  under  the  cover  slip, 
but  occasionally,  in  old  l,esions,  they 
may  be  hard,  owing  to  calcification. 

They  may  be  recognized  easily  by 
crushing  the  granules  under  the  cover 
glass,  and  examining  them,  unstained, 
with  the  microscope.  Fresh  speci- 
mens may  also  be  stained  by  Gram's. 

The  colony  as  it  appears  in  tissue  sec- 
tions or  pus  smear  consists  of  a 
rosette  arrangement.  The  central 
portion  of  the  colony  is  a  dense  mass 
of  mycelium  and  spherical  bodies. 
From  this  felted  central  mass  there 
extends  ray  or  club-like  bodies.  Club- 
shaped  enlargements  at  the  end  of 
filaments  frequently  appear  and  are 
regarded  as  a  distinguishing  charac- 
teristic of  actinomyces. 

The  organism  grows  on  a  variety  of 
media.  On  glycerine  agar  the  colonies 
develop  into  transparent  drop-like 
bodies  in  four  or  five  days  at  37°.  Old 
colonies  become  white  or  yellowish 
with  a  powdery  surface.     Some  va- 


276  BACTERIOLOGY. 


rieties  appear  distinctly  aerobic  and 
others  anaerobic.  Gelatin  is  nearly 
always  liquefied. 
In  artificial  culture  filaments  appear 
which  are  very  long  and  slender. 
They  show  true  branching,  but  have 
no  septa. 

The  young-  colony  is  a  loose  mass  of 
filaments;  older  colonies  become  dense 
and  fertile.  Rod-shaped  and  spherical 
forms  may  also  appear  and  some  fila- 
ments develop  conidia. 

Tissue  sections,  stained  with  carmine, 
followed  by  Gram's  or  Wigert's,  give 
good  results. 

Actinomycosis  -  (lumpy  jaw,  wooden 
tongue)  is  an  infectious  disease  which 
spreads  rapidly.  Cattle  are  most  com- 
monly affected,  but  humans,  horses, 
sheep  and  dogs  are  susceptible. 

The  disease  usually  runs  a  chronic 
course  and  is  distinguished  especially 
by  enlargement  of  affected  parts,  by 
hardening  of  the  tongue  and  by 
suppuration. 

Head  parts,  including  the  facial  bones, 
are  commonly  affected;  lungs  and  va- 
rious other  internal  organs  and  even 
.  the  vertibrae  may  be  involved.  The 
extent  of  injury  depends  upon  the 
location  and  the  size  of  the  involved 
area. 

There  are  several  varieties  of  acti- 
nomyces,  and  it  is  probably  specific 
in  its  relation  to  the  disease,  but  it 
is  frequently  aided  by  pus-producing 
bacteria. 

It  is,  vegetative  on  various  grasses 
especially  wild  barley,  and  infection 
occurs  by  inoculation  with  the  awns 
and  barbs  of  such  grasses  through 
the  mucous  membrane  of  the  mouth 
or  alimentary  tract.  Infection  by  in- 
halation may  occur. 

It  is  probable  that  some  special  stage 
of  development  is  necessary  either 
within  the  diseased  body  or  upon 
some  plants  in  order  that  it  may  irf- 
f^ct  animals'  bodies,  as  direct  inocu- 
lation with  pus  usually  giver 
negative  results.  Inoculation  with 
pieces  of  diseased  tissue  occasionally 
gives  positive  results. 

The  pus  scattered  over  fodder,  mangers 
and  feed  racks  probably  serves  in- 
directly as  a  source  of  dissemination. 


BACTERIOLOGY.  277 


An  active  toxin  is  evidently  not  pro- 
duced. The  disturbance  caused  by 
the  disease  is  apparently  due  to  harm- 
ful growths  in  the  tissue  and  to 
secondary  infection. 

Suppuration  is  one  of  the  conspicuous 
features,  as  is  also  the  development 
of  much  new  granulation  tissue  which 
tends  to  degenerate  at  the  center. 
Soft  organs  affected  show  a  tendency 
to  multiple  abscesses. 

Actino'bacillosis  is  probably  to  be  dis- 
tinguished from  actinomycosis.  It  is 
very  similar  in  history  and  clinical 
evidence  but  apparently  different  as 
.to  specific  cause.  The  cause  of  acti- 
nobacillosis  seems  to  be  a  bactei*ium 
found  also  in  rosette-like  masses  re- 
sembling those  of  actinomycosis. 

MYCETOMA. 
(Madura  Foat). 

This  disease  is  very  much  like  acti- 
nomycosis. It  is  more  or  less  lim- 
ited to  warmer  climates;  India,  espe- 
cially Madura.  It  consists  of  a 
chronic  productive  inflammation,  most 
frequently  attacking  the  foot,  less 
often  the  hand,  very  rarely  other 
parts  of  the  body. 

Nodular  swellings  occur,  which  break 
down  and  lead  to  abscess  formation 
and  later  to  sinuses  which  discharge 
purulent  fluid  containing  the  charac- 
teristic hard,  brittle,  black,  granular 
bodies  resembling  grains  of  gun- 
powder. The  granules  may  be  gray- 
ish white  or  yellow  and  soft  and 
grumous. 

The  appearance  of  these  granules  gives 
rise  to  two  varieties  of  the  disease: 

The  melanokL  variety  is  caused  by  a 
member  of  the  hyphomycetes  group. 

The  ochroid  variety  is  believed  by  many 
to  be  actinomycosis. 

The  bones  are  often  involved  and  a 
rarefying  osteitis  results. 

In  broth,  the  growth  is  rapid,  composed 
of  long  hyphae,  which  form  a  struc- 
ture of  a  powder  puff  appearance. 

On  agar,  at  the  end  of  a  week,  a  thick 
grayish  meshwork  of  hyphae  spread 
over  the  surface.  In  old  cultures, 
black  granules  appear  among  the 
mycelial  meshes. 


278  BACTERIOLOGY. 

On  potato  a  dense,  velvety  membrane 
appears  with  a  pale  brown  center  and 
a  white  periphery.  Brown  drops  ap- 
pear in  old  cultures. 

FATKOGENIC  MOUZkDS. 
(Hyphomycetes,  Eumycetes). 

The  relation  of  moulds  to  bacteria 
shows  them  to  occupy  a  place  higrher 
than  the  higher  bacteria  which  they 
resemble,  ii>  that  they  grow  in  fila- 
ments, but  show  in  majority  of  cases 
a  more  complicated  structure  in  pos- 
sessing a  more  distinct  wall  and  a 
definite  nucleus  and  in  their  repro- 
ductive organs.  The  hyphae  branch 
and  grow  into  a  network  called 
mycelium. 

In  the  lower  forms  each  hypha  is  a 
single  cell,  septa  only  occuring  when 
fructification  begins. 

In  the  higher  forms  the  filaments  are 
made  up  of  rows  of  cells. 

Most  forms  produce  endospores  in  a 
spore  sac  (sporangia)  situated  at  the 
end  of  a  hypha. 

Certain  varieties  have  a  primitive  sex- 
ual process,  a  conjugation  of  two 
cells  with  the  formation  of  a  zygo- 
spore, from  which  a  sporangium  car- 
rier may  arise  and  develop  a  spor- 
angium. 

Spores  may  also  be*  produced  in  so- 
called  gummae  (chlamydospores), 
which  are  swollen  portions,  segmented 
in  the  course  of  a  hypha. 

Spores  may  be  formed  as  conidia.  The 
common  molds  grow  easily  on  arti- 
ficial media  and  therefore  are  very  apt 
to  infect  the  media  during  bacteriol- 
igical  cultivations.  They  grow  espe- 
cially well  in  acid  medium  and  are 
therefore  very  often  found  on  fruit. 
The  majority  of  moulds  are  not  path- 
ogenic, but  some  are  however  true 
parasites  and  produce  a  number  of 
very  common  diseases.  Their  arti- 
ficial cultivation  is  more  diflacult  than 
the  ordinary  varieties. 

Certain  varieties  of  the  common  mucor 
has  been  reported  pathogenic  for  man 
in  that  they  have  been  found  to  pro- 
duce eye  and  ear  infections,  also  in 
a  case  of  enteritis  with  secondary 
perttonitls.  Autopsy  of  the  latter 
showed  also  multiple  abscesses  of 
brain  and  lungs. 


BACTERIOLOGY.  279 


The  aspergillus  (aspergillus  fumigatus, 
more  frequent)  is  found  more  often 
pathogenic  to  birds,  producing  a 
pseudo  tuberculosis.  Such  cases  have 
also  been  reported  in  man. 

Many  varieties  are  found  in  plant  dis- 
eases and  indirectly  may  be  of  danger 
to  man  as  when  they  form  poisonous 
substances  as  in  the  infection  of  grain 
by  claviceps  purpurea  (ergot  poison- 
ing), etc. 

The  more  common  pathogenic  forms  for 
man  are: 

Trlchopliyton  (Blngrwonn  Funsrus). 

1.  Tinea  circlnata  produces  ringworm  of 

the  body. 

2.  Tinea  tonsurans  (produces  ring- 

3.  Tinea  barbae  or        worm     of  the 

tinea  sycosis    t    hairy  parts. 
According  to  Sabourand,  there  are  two 
distinct    types    of    the    fungus,  tri- 
chophyton causing  ringworm  in  man. 

(a)  Tinea     microsporon,     with  small 

spores,  is  the  common  fungus  of 
T.  Tonsurans  of  children,  and  its 
special  seat  of  growth  is  in  the 
substance  of  the  hair.  The 
spores  are  contained  in  a  mycel- 
ium which  is  not  visible,  and 
appear  piled  up  like  zoogloea 
massess  forming  a  dense  sheath 
around  the  hair. 

(b)  Tinea    megralosporon,    with  large 

spores,  is  essentially  the  fungus 
of  ringworm  of  the  beard  and  the 
smooth  parts  of  the  skin.  The 
spores   are   always   contained  in 
distinct      mycelium  filaments, 
which    may    either   be  resistant 
when   the  hair  is  broken  up  or 
fragile   and    easily   breaking  up 
into    spores.      One-third    of  the 
cases  of  T.  Tonsurans  of  children 
are  due  to  trichophyton  megalo- 
sporon.    Cultivation  is  simple  on 
acid  glucose  agar  or  gelatine. 
Acliorion   Schoenleinii    (Favus).  This 
fungus    was    discovered   by  Schoen- 
leinii in  1839.    It  attacks  chiefly  the 
hairy  portion  of  the  body  of  man  and 
some  domestic  animals.     It  is  com- 
municated  by    contagion-.     Want  of 
cleanliness  is  a  predisposing  factor. 
The  disease  Is  extremely  chronic  and 
very  resistant  to  treatment. 


280  BACTERIOLOGY. 


In  man,  it  is  found  most  frequently  en 
the  scalp  of  persons  in  weak  health, 
especially  from  phthisis,  and  in 
undernourished  children  upon  the 
scalp.  Other  portions  of  the  skin  may 
also  be  involved.  Pathologically,  the 
disease  represents  the  reaction  of  the 
tissues  to  the  irritation  caused  by 
the  growth  of  the  fungus.  The 
spores  invade  the  hair  follicles.  The 
fungus  grows  in  the  epidermis,  the 
density  of  the  growth  causes  pres- 
sure on  the  parts  below,  lowering  the 
vitality  of  the  hair. 

The  disease  first  appears  as  a  small 
sulphur-yellow  disc  (scutulum), 
pierced  by  a  hair,  which  lesion  is 
characteristic.  It  is  readily  culti- 
vated on  artificial  media. 

Kaposi  has  reported  a  case  of  confluent 
favus  in  which  patients  died  with 
symptoms  of  severe  gastrointestinal 
irritation.  The  presence  of  the  fungus 
in  the  stomach  and  intestines  was 
demonstrated  at  autopsy. 

Microsporou  furfur  (Fityriasles  Versi- 
color). This  organism  was  discov- 
ered by  Eichstedt  in  1846,  invades 
only  the  most  superficial  layers  of 
the  skin  and  produces  the  disease 
(Chiefly  in  those  living  under  condi- 
tions of  uncleanliness,  or  among 
those  who  combine  a  tendency  to  pro- 
fuse perspiration  and  uncleanliness. 
The  organism  does  not  give  rise  to 
any  considerable  pathological  changes 
in  the  skin  or  hair. 

The  organism  shows  preference  for 
locations  such  as  the  chest,  abdomen, 
back,  and  axillae,  less  frequently  neck 
and  arms,  exceptionally  it  attacks  the 
face.  It  appears  as  scattered  spots 
of  a  color  which  varies  from  cream- 
coffee  to  reddish-brown. 

Soor  Fimgrus:  Oidium  Albicans  (Thrush). 
This  organism  was  described  by 
Langenbeck  in  1839.  It  produces  a 
disease  of  the  oral  mucous  membrane 
of  infants  during  the  early  weeks  of 
life.  It  occurs  most  frequently  in 
children  suffering  from  malnutrition. 
It  has  been  found  as  a  slight  mycosis 
in  the  vagina  of  women  and  in  rare 
cases  attacks  adults,  especially  those 
whose  health  has  been  undermined 
by  diseases,  such  as  diabetes,  typhoid, 
etc.    A  few  cases  have  been  reported 


BACTERIOLOGY.  281 


in  which  the  fungus  was  isolated 
from  abscesses  of  the  lung,  spleen, 
kidney  and  brain.  It  can  readily  be 
cultivated  in  the  ordinary  media  of 
either  acid  or  alkaline  reaction. 

The  oidium  albicans  appears  both  as  a 
yeast  and  a  mycelium,  and  therefore 
seems  to  occupy  a  position  between 
the  true  moulds  and  the  yeasts.  It 
grows  at  times  to  long  threads,  and 
under  certain  conditions,  almost  ex- 
clusively, it  will  multiply  by  budding. 
THX:  PATHOGENIC  YEASTS. 
(Blastomy  cetes) . 

These  organisms  have  been  of  great 
importance  in  brewing  and  baking 
and  recently  have  been  reported  to 
have  caused  infections  in  man  and 
animals. 

The  position  which  the  yeast  occupies 
in  systematic  biology  has  as  yet  not 
been  accurately  determined.  Their 
chief  characteristic  is  in  their 
method  of  reproduction  by  budding. 
Yeasts  can  at  times  develop  short 
hyphae  and  in  rare  cases  reproduce 
by  segmentation. 

The  most  important  property  of  yeasts 
is  that  of  producing  alcoholic  fer- 
m.entation,  and  has  been  studied  ex- 
tensively along  this  line.  The  work 
of  Pasteur  and  Hansen  along  these 
lines  developed  the  fact  that  differ- 
ences in  the  flavors  and  other  qual- 
ities of  beer,  wine,  etc.,  were  de- 
pendent upon  the  particular  species 
of  yeast  employed  for  the  fermenta- 
tion. The  fermentative  property  is 
produced  by  an  enzyme  known  as 
"zymase,"  which  transforms  sugar 
into  ethyl  alcohol.  Various  yeasts  also 
produce  other  ferments  which  split 
higher  carbohydrates  (saccharose, 
maltose,  starch)  and  prepare  them 
for  action  of  the  zymase. 

The  yeasts  employed  in  practice  are 
spoken  of  as  "culture  yeasts"  and 
those  which  act  as  disturbing  factors 
in  fermentation  are  called  "wild 
yeasts";  the  latter  usually  producing 
only  a  slight  degree  of  fermentation. 
The  culture  yeast  cell  is  oval  or  ellip- 
tical in  shape,  while  the  wild  species 
are  more  often  round  or  globular  and 
known  as  "torula"  forms.  Sausage- 
shaped  and  thread  forms  are  also 
found. 


282  BACTERIOLOGY. 

The  individual  cell  is  highly  refractive 
and  varies  greatly  in  size  even  in 
those  of  the  same  species  or  the  same 
culture.  The  cell  contains  a  nucleus, 
which  can  be  demonstrated  by  stain- 
ing. During  the  process  of  budding 
the  nucleus  moves  toward  the  peri- 
phery and  divides,  the  limiting  mem- 
brane of  the  cell  ruptures  or  a  pro- 
trusion develops  (daughter  cell),  this 
rapidly  increases  in^  size  and  assumes 
the  shape  of  the  mother  cell. 

Spore  formation  takes  place  in  the 
yeast,  which  is  of  importance  in  th'e 
continuation  of  the  species  and  also 
for  propagation.  The  nucleus  divides 
into  several  fragments,  each  of  which 
becomes  the  center  of  a  new  cell. 
These  new  cells  (within  the  original 
cell)  possess  a  firm  membrane,  a  cell 
nucleus  and  a  little  dense  protoplasm. 
As  a  rule  one  cell  does  not  produce 
more  than  four  spores,  called  "astro- 
spores."  The  number  of  spores 
formed  varies,  however,  but  is  con- 
stant for  a  species. 

The  Pathogenic  forms  are: 

Saccharomyces  Busse,  isolated  by  Busse 
in  1894  from  a  woman's  tibia.  Au- 
topsy showed  broken  down  nodules 
an  several  bones,  in  the  lungs  and 
kidney. 

Saccharomyces  tumefaciens,  isolated  by 
Curtis  in  1895  from  multiple  tumors 
on  the  hips  and  neck  which  resembled 
microscopically  softened  myxosar- 
coma. 

This  yeast  is  pathogenic  for  mice,  rats 
and  dogs.  In  generalized  blastomyco- 
sis, the  lung  seems  to  be  the  seat  of 
primary  infection. 

Cases  described  by  Rixford  and  Gil- 
christ as  coccidiosis  (thought  to  be 
a  protozoan  disease)  were  unquestion- 
ably due  to  blastomycetes.  Fontaine, 
Hasse  and  Mitchell  reported  a  typical 
case  of  systematic  blastomycosis. 
Lundogaard  reported  a  case  of  optlial- 
mia  due  to  a  yeast.  Tokishige  re- 
ported an  epidemic  of  ulcerous  skin 
diseases  among  horses  in  Japan  to  be 
due  to  one  of  the  saccharomyces. 
Kartulis  described  100  cases  of  a  skin 
affection  occuring  in  the  gluteal  re- 
gions of  men  from  which  he  isolated 
the  ordinarj'-  saccharomyces  cere- 
visiae.      Kessler     reported     a  skin 


BACTERIOLOGY.  283 

lesion  in  an  infant  as  due  to  a  blas- 
tomycete. 

Attempts  have  been  made  to  connect 
the  development  of  cancerous  growth 
with  blastomycetes  by  reason  of  the 
similarity  between  the  yeasts  and  the 
inclusions  or  so-called  parasites  of 
cancer  and  also  by  the  fact  that 
yeasts  will,  when  injected  into  the 
animal  body,  produce  tumor-like 
nodules.  These  masses  are  not 
tumors  in  a  patholog-lc  sense,  but 
merely  masses  of  yeast  cells  mixed 
with  inflammatory  tissue  prolifera- 
tion. 

DISEASES  OF  UNKNOWN  ETIOZ^OGY. 

Measles.  Many  bacteria  as  well  as  sup- 
posed protozoan  bodies  have  been  de- 
scribed by  various  investigators  as 
occuring  on  the  mucous  membrane  or 
in  the  blood  of  those  sick  with  the 
disease. 

■  Home,  in  1759,  claimed  to  have  pro- 
duced measles  of  a  modified  and 
milder  type  by  rubbing-  cotton  swabs 
saturated  with  the  blood  of  patients 
affected  with  measles  on  wounds 
made  on  the  arms  of  other  individ- 
uals. It  is  not  certain  that  he  pro- 
duced the  disease  at  all. 

Positive  results  in  experimental  inocu- 
lation have  been  reported  by  Stewart 
(1799),  Speranza  (1822),  Katowa 
(1842),  and  Nigirr  (1850).  These  re- 
sults are,  however,  not  satisfactory. 

Heckton,  in  1905,  produced  the  disease 
experimentally  by  injecting  the  blood 
of  a  measle  patient,  during  the  fourth 
day  of  the  disease,  into  two  students. 

Attempts  at  cultivation  were  negative, 
but  the  virus  of  measles  will  live 
for  at  least  24  hours  when  mixed  with 
acitic  broth. 

Scarlet  Pever  is  an  acute,  febrile,  highly 
infectious  disease  characterized  by  a 
diffuse  punctate  erythematous  skin 
eruption  accompanied  by  catarrhal, 
croupous  or  gangrenous  inflammation 
of  the  upper  respiratory  tract  and  by 
manifestations  of  general  systemic 
infection.  Both  streptococci  and  pro- 
tozoa have  been  described  as  the 
etiological  factor  in  the  disease. 

Crooke  in  1885  demonstrated  strepto- 
cocci in  the  cadavers  of  scarlet  fever 
victims. 


284 


BACTERIOLOGY. 


Babinsky  and  Sommerfield  in  1900  re- 
ported the  presence  of  streptococci  in 
the  heart's  blood  Of  eight  rapidly  fatal 
cases  of  scarlet  fever. 

Mallory  in  1904  described  protozoan 
bodies  found  in  the  skin  of  four 
scarletina  cases.  The  bodies  described 
and  found  by  him  between  the  epithe- 
lial cells  were  small  and  not  unlike 
the  Plasmodium  of  malaria.  They 
stain  easily  by  methylene  blue. 

The  bodies  are  still  under  investigation. 
Field  and  others  have  failed  to  dem- 
onstrate. The  streptococci  are,  how- 
ever, certainly  present,  but  are  con- 
sidered secondary  invaders,  and  by 
reason  of  this  fact  Moser  adopted  the 
use  of  antistreptococcic  serum  and 
claims  exceptional  results. 

Typhus  Pever.  This  is  an  infectious 
disease  of  a  five-day  or  more  incuba- 
tion period  and  characterized  by  high 
temperature  and  a  petechial  rash. 
The  etiologrical  factor  has  not  been 
definitely  determined. 

Nicolle  in  1909  transmitted  the  disease 
to  the  chimpanzee,  and  from  this  to 
the  macacus  with  typical  eruption  in 
each  case.  He  was  not  able  to  trans- 
mit the  disease  from  monkey  to 
monkey. 

Anderson  and  Goldberger  in  1909  trans- 
mitted the  typhus  fever  of  Mexico 
(kabardillo)  directly  from  the  human 
being  to  the  macacus  and  capuchin. 

Ricketts  and  Walker  in  1910  from  re- 
searches came  to  the  following  con- 
clusions: 

1.  M.    rhesus    can    be    infected  by 

injecting  the  blood  of  man  dur- 
ing the  8  to  10-day  period  of  the 
fever. 

2.  It  could  not  be  transmitted  from 

monkey  to  monkey. 

3.  The  disease  may  produce  so  mild 

symptoms  in  the  monkey  as  to 
be  unrecognized  clinically,  vac- 
cination results. 

4.  Immunity  test  is  proof  of  pre- 

vious occurrence  or  nonoccur- 
rence of  the  disease  within  a 
period  of  one  month. 

5.  It  is  transmitted  to  the  monkey 

by  the  bite  of  the  louse. 


BACTERIOLOGY.  285 


6.  A    monkey    was    infected  with 

typhus  by  introducing  feces 
and  abdominal  contents  of  in- 
fected lice  into  small  incisions. 

7.  The  blood  of  patients  taken  from 

the  7th  to  12th  day  and  stained 
(Giemsa)  will  show  bacilli  of 
the  hemorrhagic  septicaemic 
group  morphology. 

8.  No   cultures   could   be  obtained, 

but  fresh  preparations  showed 
forms  like  those  above  with- 
out motility. 

9.  Dejecta   of   lice    were  examined 

and  the  organism  found  in  the 
infected  lice  and  occasionally 
in  noninfected  ones. 

Plotz  has  recently  reported  the  cultiva- 
tion of  a  gram  positive  pleomorphic 
anaerobic  organism  from  the  blood  of 
cases  of  Brillo  disease  and  also  from 
typhus  cases.  Complement  fixation 
was  obtained  when  this  organism  was 
used  as  antigen. 

Smallpox,  or  variola,  is  an  acute  infec- 
tious disease  characterized  by  an  epi- 
dermic eruption  of  macules,  vesicles 
and  pustules,  which  upon  healing  pro- 
duce cicatrices  of  varying  extent  and 
depth. 

The  disease  was  first  described  by 
Phozes  in  the  tenth  century.  It  may 
have  developed  first  in  certain  re- 
gions of  Asia  and  central  Africa. 

Severe  epidemics  have  swept  China  and 
Eastern  countries  many  centuries 
before  Christ,  also  Europe,  especially 
at  the  time  of  the  Crusades. 

The  disease  was  widespread  when 
Jenner  (1798)  showed  conclusively 
that  vaccination  with  cowpox  afforded 
protection. 

The  etiological  factor  is  as  yet  not 
determined.  Streptococci,  often  found 
in  the  vesicles  and  pustules  and  con- 
tribute materially  to  the  fatal  out- 
•come  of  the  disease,  are  secondary 
invaders. 

Guarneiri  (1892)  named  certain  inclu- 
sions present  in  the  epithelial  cells  of 
smallpox  lesions  in  a  rabbit's  cornea, 

"Cytorrhyctes  variola,"  and  believed 
them  to  be  protozoa. 

Councilman  believed  the  bodies  (vaccine 
bodies)  to  be  protozoa,  and  describes 
two  cycles  in  its  development,  one 
intracellular  and   the   other  intranu- 


286  BACTERIOLOGY, 


clear,  and  that  intranuclear  infection 
occurs  only  in  smallpox. 

Calkins,  working  with  Councilman,  also 
believes  them  to  be  protozoa  of  the 
class  rhizopoda. 

Ewing  admits  that  the  vaccine  bodies 
are  probably  specific  for  variola,  but 
calls  attention  to  inclusions  found  in 
diphtheria,  measles,  g-landers  and 
other  infectious  processes  which  can- 
not be  considered  as  etiological  fac- 
tors in  these  diseases.  He  believes 
that  all  forms  so  far  described  are 
degeneration  products,  some  specific, 
others  not.  The  similarity  of  cow- 
pox  (vaccinia)  and  small  pox  has 
been  the  subject  of  controversy.  Many 
observers  claim  that  although  related 
to  each  other  they  are  essentially 
different. 

Others  maintain,  and  this  seems  to  be 
the  prevailing  opinion,  that  cowpox 
or  vaccinia,  when  inoculated  into  man, 
represents  the  altered  and  attenuated 
variety  of  variola.  It  has  been  claim- 
ed that  cowpox  was  originally  trans- 
mitted to  cattle  by  human  beings 
affected  with  smallpox.  The  immun- 
ity caused  by  successful  vaccination 
is  not  permanent  and  varies  in  its 
juration  in  different  individuals.  Al- 
though immunity  may  last  for  10  to 
15  years  it  is  well  to  be  vaccinated 
every  year,  if  exposed  to  the  disease. 
If  the  vaccination  is  unnecessary  it 
will  not  be  successful.  (See  small- 
pox vaccine.) 

Babies  (Hydrophobia)  is  an  acute  in- 
fectious disease  of  mammals,  depend- 
ent upon  its  specific  virus  and  com- 
municated to  susceptible  animals  by 
the  saliva  of  an  infected  animal  com- 
ing in  contact  with  a  broken  surface, 
usually  through  a  bite.  No  bacteria 
have  been  discovered  that  are  con- 
sidered as  factors  in  the  disease.  In 
1903,  Negri  described  peculiar  svriic- 
tures  which  he  observed  in  the  cell  of 
the  central  nervous  system  of  rabid 
animals,  which  he  claims  are  not  only 
specific  for  hydrophobia  but  are  prob- 
ably animal  parasites  and  cause  the 
disease.  His  later  studies  confirm 
his  previous  work  and,  so  far  as  the 
diagnostic  value  of  these  bodies  is 
concerned,  he  has  been  corroborated 
by  numerous  investigators. 


BACTERIOLOGY.  287 

Williams  in  1906,  was  convinced  that 
these  cell  inclusions  were  animal  or- 
ganisms and  called  attention  to  the 
similarity  between  their  structures 
and  that  of  the  rhizopoda.  He  gave 
them  the  name  Neurorycetes  Hydro- 
phobia. 

It  is  not  possible,  at  the  present  time, 
to  decide  absolutely  whether  or  not 
the  Negri  bodies  should  be  regarded 
as  parasites  or  specific  degeneration 
products. 

The  virus  of  rabies  has  been  shown  to 
be  partially  filterable  through  course 
Berkefeld  filters.  The  retained  por- 
tion is  always  more  virulent  than  the 
filtrate.  This  would  seem  to  indi- 
cate that  there  are  some  forms  just 

^  within  the  limit  of  visibility  and 
others  larger  which  correspond  with 
what  "we  know  of  the  variation  in  size 
of  the  Negri  bodies. 

The  largest  forms  of  Negri  bodies  are 
about  18  microns  and  the  smallest 
about  0.5  micron.  They  are  round, 
oval,  oblong,  triangular  or  ameboid. 
They  show  a  hyalene-like  cytoplasm 
with  an  entire  margin  containing  one 
or  more  chromatin  bodies  which  have 
a  more  or  less  complicated  and  reg- 
ular arrangement. 

The  demonstration  of  Negri  bodies  in 
tissues  is  carried  out  by  procuring  a 
small  piece  of  tissue  from  the  cere- 
bellum or  from  the  center  of  the  hip- 
pocampus major  and  fixing  it  for  12 
hours  in  Zenker's  fluid.  It  is  then 
washed  in  water  and  dehydrated  in 
graded  alcohol,  embedded  in  paraffin 
and  sectioned.  The  sections  are  at- 
tached to  slides  and  placed  in  the  fol- 
lowing solution  from  12  to  24  hours: 
Methylene-blue    (Grueber    00)  one 


per  cent  35  cc. 

Eosin   (Gruebler  BA)   one  per  cent 

 35  cc 

Distilled  water  100  cc. 

Then  differentiate  in: 

Absolute   alcohol  30  cc. 

Sodium  hydrate,  1  per  cent  in  ab- 
solute alcohol  5  cc. 


Allow  them  to  remain  for  about  five 
minutes,  wash  in  absolute  alcohol 
then  place  in  water,  then  in  water 
slightly  acidified  with  acetic  acid. 
Dehydrate  in  absolute  alcohol,  clear 
in  xylol  and  examine. 


288  BACTERIOLOGY. 


The  nerve  cells  are  stained  pale  blue 
and  in  their  cytoplasm  close  to  the 
nucleus  or  near  the  axis-cylinder  pro- 
cess are  seen  oval  bodies  stained  a 
deep  pink.  They  show  a  more  deeply 
stained  periphery  than  the  interior 
which  often  contains  small  vacuole- 
like  bodies.  There  may  be  one,  three 
of  four  in  a  single  cell. 

The  Negri  bodies  may  be  rapidly  dem- 
onstrated in  smears  of  brain  tissues 
for  diagnostic  purposes  as  follows: 

A  small  pin  head  sized  piece  of  brain 
tissue  taken  from  the  cerebellum  or 
the  center  of  the  hippocampus  major 
is  placed  on  one  end  of  the  slide 
under  a  cover  glass  and  then  gently 
squeezed  until  the  tissue  is  flattened 
out  into  a  thin  layer.  The  glass  cover 
is  then  gently  slipped  across  the  slide 
so  as  to  smear  the  brain  tissue  along 
the  entire  surface.  These  smears  are 
fixed  in  methyl  alcohol  and  stained 
by  the  Giemsa  method.  The  bodies 
are  stained  light  blue  in  contrast  to 
the  darker  and  more  violet  cell  bodies. 

The  smears  may  also  be  stained  by 
van  Gieson's  stain  as  follows: 

Fix  the  smears  in  methyl  alcohol,  wash 

.  in  water,  cover  with  the  fresh  pre- 
pared stain,  steam,  rinse  in  water  and 
dry. 

Distilled   water  10  cc. 

Saturated     alcoholic     solution  of 

rosanalin  violet   2  drops. 

Saturated      a^queous      solution  of 
methylene-blue    diluted  one-half 

 2  drops. 

The  Negri  bodies  stain  magenta;  their 
contained  granules,  blue;  the  air  cells, 
blue;  and  the  red  blood  cells,  yellow. 
Or  by  the  Williams  and  Lowden  modi- 
fication: 

Distilled  water  10  cc. 

Saturated     alcoholic     solution  of 

basic-fuchsin  3  drops. 

Loefl^er's     alkaline  methylene-blue 

 2  cc. 

The  bodies  assume  a  brilliant  hue  and 
contain  in  their  interior  darkly  stained 
irregular  particles,  probably  chro- 
matim  bodies. 
All  of  the  work  should  be  controlled  by 
careful  animal  inoculation.  The  work 
of  the  smear  method  in  diagnosis  has 


BACTERIOLOGY. 


289 


been  summarized  by  Parke  and  Wil- 
liams as  follws: 

1.  Negri    bodies    demonstrated,  diag- 

nosis rabies. 

2.  Negri   bodies   not  demonstrated  in 

fresh   brains,   very   probably  not 
rabies. 

3.  Negri  bodies  not  demonstrated  in 

decomposing   brains,  uncertain. 
.4.  Suspicious  bodies  in   fresh  brains, 
probably  rabies.     (See  rabic  vac- 
cine.) 

Whooping'  Cougrli.  (See  Bordet  and 
Gengou  Bacillus.) 

Bordet  and  Gengou  in  1906,  described  a 
bacillus    which    they     consider     the  • 
specific  organism  because  they  obtain 
with  it  the  complement  fixation  re- 
action. 

Woolstein,  1909,  was  not  able  to  cor- 
roborate their  work.  This  bacillus 
differs  slightly  from  a  bacillus  which 
differed  only  slightly  from  the  ba- 
cillus of  influenza  that  had  been  de- 
tected by  Jockman,  Krause  and  Wool- 
stein in  practically  all  cases  of 
whooping  cough  during  the  acute 
stages. 

Mumps.  Diplococci  had  been  considered 
as  possibly  being  the  exciting  or- 
ganism. 

Noma.  A  streptothrix  pseudo  diph- 
theria bacillus,  diphtheria  bacilli  are 
the  organisms  most  usually  present 
in  cases  of  noma  but  it  is,  as  yet, 
undecided  whether  the  disease  is  due 
to  one  or  to  several  microorganisms. 
A  special  predisposition  of  the  tissues 
is  necessary. 

Articular  rheumatism.  (See  Poynton 
and  Paine  diplococcus).  Most  bac- 
teriologists believe  the  exciting  factor 
has  not  yet  been  identified.  Strepto- 
cocci have  been  of  all  bacteria  most 
frequently  found  in  the  search  for  the 
etiological  factor  in  the  synovial 
fluid,  blood  vegetation  of  heart  valves, 
and  in  exudates  on  tonsils,  etc. 

The  streptococci  and  the  other  coci 
found  are  probably  important  second- 
ary infections. 

Beriberi.  Both  bacteria  and  protozoon 
microorganisms  have  been  considered 
as  exciting  factors  but  nothing  def- 
inite has  been  proven. 


290  BACTERIOLOGY. 


Pellagrra.  Some  investigators  believe 
this  to  be  due  to  a  microorganism, 
while  others  believe  it  to  be  an  in- 
toxication similar  to  that  of  ergot 
poisoning. 

THE  ui;tra  microscopic 

ORGANISMS. 

Infective  material  from  a  number  of  in- 
fectious diseases  may  with  certain 
precautions  be  passed  through  stone 
filters  of  varying  degrees  of  porosity 
and  still  reproduce  the  disease  with 
all  its  characteristic  symptoms  when 
inoculated  into  susceptible  animals. 
Microscopic  examination  of  the  fil- 
trate, except  in  one  or  two  diseases, 
does  not  show  the  faintest  sign  of 
particulate  matter. 

The  precautions  necessary  in  such  fil- 
tration are: 

1.  A  perfect  filter,  which  will  abso- 

lutely retain  all  known  bacteria, 
allowing  none  to  pass  into  the 
filtrate. 

2.  The   filtration  must   be  completed 

within  a  moderate  time  by  reason 
of  the  fact  that  bacteria  may,  in 
a  media  which  contains  a  certain 
amount  of  albuminous  material, 
grow  through  the  filter. 

3.  The  material  to  be  filtered  should 

be  greatly  diluted  and  first  passed 
through    filter    paper    so    as  to 
avoid  the  clogging  action  of  ex- 
traneous material. 
If.  with  these  precautions,  the  filtrate 
is  pathogenic,  ascertain  whether  the 
symptoms  are  due  to  the  microorgan- 
isms and  not  to  a  toxin.     This  may 
be  determined  with  almost  absolute 
certainty  by  inoculating  a  series  of 
animals  successively  with  the  filtrate 
obtained  from  a  previously  so  inocu- 
lated animal. 
Anterior  Poliomyelitis.   Landsteiner  and 
Popper    thought    the    virus  belonged 
to    the    class    of    invisible  protozoa. 
They  were  able  to  transmit  the  dis- 
ease to  apes.     They  made  intraperi- 
toneal  inoculations  with  spinal  cord 
and  produced  typical  symptoms  and 
lesions.     They  were  unable  to  trans- 
mit the  disease  from  ape  to  ape. 
Flexner   transmitted   the   disease  from 
monkey  to  monkey  by  intracerebral 
Inoculations.      Landsteiner    and  Le- 


BACTERIOLOGY.  291 


vaditi  transmitted  the  disease  from 
monkey  to  monkey  and  found  that  the 
virus  lived  four  days  outside  of  the 
body.  They  found  the  virus  in  the 
salivary  glands  and  suggested  the 
moist  or  dry  saliva  as  a  source  of 
contagion.  Plexner  transmitted  the 
disease  by  means  of  inoculations  into 
the  blood  or  peritoneal  cavity,  also  by 
subcutaneous  inoculation,  and  found 
the  virus  to  be  filterable. 

The  virus  has  been  shown  to  be  pre- 
servable  under  glycerin  for  ten  days 
and  retains  its  virulence  to  from  7  to 
11  days,  when  dried.  The  virus  will 
remain  active  when  frozen  for  a 
period  of  40  days,  but  is  extremely 
sensitive  to  heat,  being  destroyed  by 
a  temperature  of  45°  to  50°  C.  main- 
tained for  30  minutes.  Flexner  and 
Noguchi  placed  small  bits  of  an  emul- 
sion of  the  brain  of  monkeys  dead  of 
poliomyelitis  under  conditions  similar 
to  the  cultivation  of  the  Treponema 
pallidum.  Noguchi  found  after  five 
days  that  an  opalescence  appeared, 
which  increased  until  the  tenth  day 
when  sedimentation  began.  Micro- 
scopical examination  by  Giemsa's  re- 
vealed small  ovoid  bodies  arranged  in 
pairs,  short  chains  and  masses.  Sim- 
ilar bodies  were  later  found  in  poli- 
omyelitis tissue.  Cultures  were  ob- 
tained from  glycerinated  virus,  fresh 
virus  and  from  filtered  and  infiltered 
material.  When  these  cultures  were 
injected  into  monkeys,  typical  lesions 
and  death  were  produced  up  to  the 
eighteenth  generation  of  cultivation 
on  artificial  media. 

Foot  and  Mouth  Disease.  This  is  a 
highly  infectious  disease  occurring 
chiefly  in  cattle,  sheep  and  goats, 
more  rarely  in  other  domestic  an- 
imals. It  appears  as  a  vesicular 
eruption  upon  the  mucus  membrane 
of  the  mouth  and  upon  the  delicate 
skin  between  the  hoofs.  Usually  the 
disease  is  mild.  The  vesicles  form 
small  ulcers'  and  pustules  which 
gradually  heal  with  a  disappearance 
of  systemic  symptoms.  The  disease 
may  be  complicated  by  a  gastro- 
intestinal or  pulmonary  infection  and 
may  end  in  death.  The  disease  is 
generally  transmitted  from  animal  to 
animal  by  means  of  the  virus  con- 


292  BACTERIOLOGY. 


tained  in  the  vesicles.  On  rare  oc- 
casions, the  disease  is  transmitted  to 
man,  usually  by  direct  contact  or  by 
drinking  the  milk  of  animals  suffer- 
ing from  the  disease.  The  course  of 
the  disease  in  man  is  usually  very 
mild. 

Loeffler  and  Frosch  in  1898,  diluted  the 
contents  of  an  unbroken  vesicle  with 
20  to  40  times  its  volume  of  water, 
passed  the  solution  through  a  Berke- 
feld  filter  and  found  that  the  filtrate 
remains  infectious  for  some  time. 
The  virus  of  the  disease  is  readily 
destroyed  by  heating  to  60°  G.  Loef- 
fler has  actively  immunized  horses 
and  cattle  with  greater  doses  of  the 
virus  obtained  from  vesicles  and  with 
the  sera  of  such  animals  has  produced 
passive  immunity.  One  attack  of  foT)t 
and  mouth  disease  protects  against 
subsequent  attacks.  This  immunity 
may  last  for  years,  but  a  case  of  re- 
currence within  a  single  year  has 
been  reported. 

Yellow  Pever  is  an  acute  infectious  dis- 
ease which  prevails  endemically  in 
the  tropical  countries  with  no  char- 
acteristic lesion  except  jaundice  and 
hemorrhage.  Other  lesions  that  ex- 
ist are  those  common  to  toxemia. 

Sanarelli,  1897,  found  the  bacillus 
icteroid  circulating  in  the  blood  and 
in  the  tissues  of  most  yellow  fever 
patients  and  it  was  thought  by  many 
to  be  the  causative  organism,  but  has 
been  rejected.  The  U.  S.  Army  Com- 
mission (Reed,  Carroll,  Agramonte 
and  Lazear)  established  the  fact  that 
the  disease  was  carried  from  one  in- 
fected person  to  another  through  the 
agency  to  the  mosquito.  This  Com- 
mission established  the  following 
facts: 

1.  Yellow  fever  is  transmitted  under 

natural  conditions  only  by  the 
bite  of  a  mosquito  (Stegomyia 
calopus)  that  at  least  12  days 
before  had  fed  upon  the  blood 
of  the  patient,  sick  with  this  dis- 
ease during  the  first  three  days 
of  his  illness. 

2.  Yellow  fever  can  be  produced  arti- 

ficially by  subcutaneous  injection 
of  blood  of  a  person  sick  with 
this  disease  during  the  first  three 
days  of  his  illness. 


BACTERIOLOGY.  293 


3.  Yellow  fever  is  not   conveyed  by 

fomites. 

4.  The  bacillus  icteroid  has  no  causa- 

tive relation  to  yelow  fever. 
Although  the  specific  parasite  has  not 
yet    been    discovered,    the  following 
facts  have  been  brought  out: 

1.  The  causative  agent  requires  two 

hosts   for   the   completion   of  its 
cycle.     (A  mammal  and  arthro- 
\  pod.) 

2.  There   is   a  definite   time  between 

the  bite  of  a  mosquito  and  the 
infectivity  of  the  blood  (average 
5^  days)  and  a  definite  time  that 
the  blood  remainl^  infective  (three 
days.) 

3.  The   blood   after   passing  through 

the  finest  porcelain  filters  remains 
infective  during  these  three  days. 

4.  The  blood  will  lose  its  virulence  in 

48  hours  when  exposed  to  the  air 
and  at  a  temperature  of  24°  to 
30^  C.  If  protected  from  the  air 
by  oil,  at  the  same  temperature, 
it  remains  virulent  for  from  5  to 
8  days.  It  becomes  non-virulent 
if  heated  at  55**  C.  for  five 
minutes. 

5.  The  bite  of  an  infected  mosquito 

does  not  become  infectious  until 
12    days    (at    a    temperature  of 
31°   C.)    after   it   has   bitten  the 
first  patient. 
The  infective  blood  filtrates  show  noth- 
ing with  dark  field  illumination,  ex- 
cept small  motile  granules  similar  to 
those  found  in  healthy  persons. 
The  necessity  for  a  second  host  and  the 
long  incubation  time  required  before 
the  host  becomes  infected,  after  biting 
a  yellow  fever  patient,  seem  to  point 
to  a  protozoan  organism  as  a  causa- 
tive factor. 
Dengue,   Ashburn   and   Craig   claim  to 
have  reproduced  the  disease  in  sus- 
ceptible individuals  along  the  lines  of 
procedure  employed  in  yellow  fever. 
The  intermediate  host  in  natural  in- 
fection    they    claim     to     be  culex 
fatigans. 

South  African  Horse  Sickness  occurs  in 
warm  weather,  as  a  rule,  and  seems 
to  be  more  common  in  animals  which 
pass  the  night  outside.  The  disease 
manifests  itself  by  uneasiness,  diflfi- 
culty  in  breathing,  and  the  appearance 


294  BACTERIOLOGY. 


of  reddish  froth  from  the  mouth. 
The  temperature  rises  in  the  daytime, 
but  drops  at  night.  Edematous  swell- 
ing of  the  head  and  neck  may  appear 
in  severe  cases. 
Contagrious  FlenropneTunonia  of  Cattle. 
This  disease  does  not  affect  other 
species.  It  appears  as  inflammation 
of  the  lung's  and  pleura  with  necrosis. 
Nocard  and  Roux  have  cultivated  an 
organism  in  collodion  sacs  placed  in 
the  peritoneal  cavity  of  a  rabbit, 
using  a  mixture  of  serum  and  bouil- 
lon. After  two  weeks  a  very  faint 
turbidity  appears  in  the  sacs,  coin- 
cidently  the  fluid  becomes  infected. 
The  causative  factor  in  this  disease 
has  been  made  to  grow  and  produce 
disease  in  new  animals  and,  as  at  the 
highest  limit  or  present  magnifica- 
tion, it  is  seen  to  consist  of  minute 
granules. 

Rinderpest.  A  fatal  European  and 
African  disease  of  cattle  is  character- 
ized by  inflammation  of  the  intestinal 
mucous  membrane.  No  organism  can 
be  seen. 

Trachoma  is  a  disease  of  the  eye  which 
is  characterized  by  a  progressive  fol- 

»  licular  inflammation  of  the  conjunc- 
tiva followed  by  cicatrization. 

Prowazek  (1907)  announced  the  dis- 
covery of  small  organisms  the  cause 
of  the  disease  and  named  them 
Chalamydozoa  and  believes  they  oc- 
cupy a  place  between  bacteria  and 
protozoa. 

The  organism  is  found  only  in  the 
early  acute  cases. 

Prowazek  states  that  the  organism 
grows  in  a  characteristic  manner  in 
the  conjunctival  epithelial  cells.  It  is 
so  small  that  it  cannot  at  first  be 
seen,  only  the  mantle  can  be  demon- 
strated, which  stains  blue  with 
Giemsa.  The  organism  appears  as  a 
small  red  granule  within  the  blue 
body.  As  the  organisms  increase  in 
size  and  numbers  the  blue  mantles 
disappear,  leaving  a  mass  of  small, 
round,  or  slightly  elongated  red 
bodies. 

Lipschutz  points  out  the  fact  that 
dhalamydozoa,  although  visible,  pass 
through  filters,  and  with  Borrel 
claims  to  have  discovered  a  similar 
organism  in  Molluscum  contagiosum 


BACTERIOLOGY.  295 

of  man  and  birds.  He  also  believes 
that  the  Volpino's  motile  granules 
discovered  in  vaccinia  by  the  ultrami- 
croscpoe  and  his  own  bodies  of  rabies 
belong  to  the  same  class. 
By  reason  of  their  round  form  he  sug- 
gested the  name  "Strongyloplasmen." 

SFIBbCHETAE  AND  Al^I^IES. 

The  microorganism  known  as  spirocheta 
(name  introduced  by  Ehrenberg  in 
1838,  who  differentiated  it  from 
spirillum  by  its  flexibility)  are  slender, 
undulating,  cork-screw  like  threads 
which  vary  both  structurally  and  cul- 
turally from  bacteria.  .  The  organisms 
were  formerly  regarded  as  bacteria, 
belonging  to  the  general  group  of  the 
spirillum.  Schaudinn,  by  a  careful 
morphological  study,  claims  that 
many  of  these  forms  are  protozoa. 
Other  observers  have  not  agreed  with 
him. 

The  reason  for  considering  these  or- 
ganisms as  protozoa  are: 

1.  Their  flexibility  and  the  indication 

in  many  of  longtitudinal  division 
and  of  undulating  membrane. 

2.  The  demonstration  of  forms  inter- 

mediate betwefen  trypanosomes 
and  the  spirochetes  (SP.  bal- 
bianii). 

3.  The   spirochetal   forms   of  certain 

trypanosomes  (TR.  Noctuae.) 
The  reasons  for  favoring  the  bacterial 
nature  of  spirochetes  are; 

1.  The  rigidity   of  some  forms,  the 

lack,  of  undulating  membrane  in 
most  and  of  definite  nuclei  ap- 
paratus in  all,  the  evidence  of 
transverse  division  of  all  and  of 
flagella  arising  from  the  blepharo- 
plast  in  some. 

2.  The   cultivation   of   certain  forms 

are  the   SP.   refringens  and  the 
SP.  Obermeieri  for  many  gener- 
ations  '-without    development  of 
trypanosome  forms. 
It  is  probable  that  the  spirochetes 
and  their  allies   occupy   a  posi- 
tion   intermediate    between  the 
protozoa  and  bacteria. 
Spirochetes  of  the  Month. 
I  Non-pathogenic  forms  commonly  found 
in  normal  mouths  are: 


296  BACTERIOLOGY. 


1.  Spirocheta   buccalis    has    three  to 

ten  irregular  flat  coils.  No  true 
cilia  have  been  demonstrated. 
Some  authorities  claim  for  it  an 
undulating-  membrane.  It  stains 
violet  with  Giemsa. 

2.  Spiroclieta  Dentium  is  much  smaller 

than  the  buccalis  and  somewhat 
similar  to  the  pallidum  in  stain- 
ing qualities  and  fixity  of  its 
coils  when  in  motion.  It  stains 
with  Loeffier's  flagella  stain.  A 
flagellum  is  present,  but  no  un- 
dulating membrane  or  nuclear 
material  has  been  demonstrated. 
Spirals  are  numbered  from  four 
to  twenty.  This  organism  has 
been  cultivated. 

3.  A  form  which  seems  to  occupy  a 

position  between  the  two  men- 
tioned above  has  been  found  in 
the  mouth,  but  has  less  regular 
spirals. 

Spiroclieta  refrlngrens  is  an  organism 
that  is  found  in  the  mouth.  It  is  also 
frequently  associated  with  triponema 
pallidum  in  the  various  lesions  of 
syphilis,  with  which  it  is  probably  a 
secondary    invader.      The  irregular, 

»  wide,  flat  spirals  number  from  three 
to  fifteen  and  change  their  shape  dur- 
ing motion.  Stains  easily  and  quick- 
ly with  Giemsa. 

Schaudinn  stated  that  it  possesses  an 
undulating  membrane.  A  terminal 
cilia  has  been  demonstrated  by 
Levaditi,  who  also  cultivated  the  or- 
ganism in  collodion  sacs  in  the  peri- 
toneal cavity  of  a  rabbit. 

Spiroclieta  Vincentl  (Vincent's  angfina). 
Vincent's  angina  consists  of  an  in- 
flammatory lesion  in  the  mouth,  phar- 
ynx or  throat,  situated  most  fre- 
quently on  the  tonsils,  beginning  as 
an  acute  stomatitis,  pharyngitis,  or 
tonsilitis,  which  soon  leads  to  the 
formation  of  the  pseudo-membrane 
closely  resembling  that  caused  by  the 
bacillus  of  diphtheria.  This  may  be 
followed  by  distinct  ulcers  with  a 
well  defined  margin  and  punched  out 
appearance.  The  disease  is  usually 
mild,  but  occasionally  moderate  fever 
and  systemic  disturbances  are  noted. 
Vincent  described  the  presence  of  two 
organism  as  causative  agents  of  the 
disease;    the    one    a    large  spindle- 


BACTERIOLOGY.  297 


shaped,  or  fusiform  bacillus;  the 
other,  a  spirocheta  similar  to  the 
"middle  form"  found  in  the  mouth. 
By  reason  of  the  fact  that  th^e  two 
organisms  were  always  found  to- 
gether, they  were  at  first  believed  to 
represent  two  forms  which  lived  in 
symbiosis.  Tunnicliff  believes  that 
these  two  forms  merely  represent  dif- 
ferent stages  of  development  of  the 
same  organism. 

The  bacilli  vary  in  length,  thick  at  the 
center,  from  which  they  taper  gradual- 
ly towards  the  ends,  and  end  in  blunt 
or  sharp  points.  The  organism  is 
usually  straight,  though  sometimes  it 
may  be  slightly  curved.  They  stain 
with  the  stronger  aniline  dyes  and 
usually  decolorize  by  Gram's.  They 
stain  more  deeply  near  the  end  and 
show  a  banded  or  striped  alternation 
of  stain  and  unstained  areas  in  the 
central  bodies. 

The  spirilla  are  usually  somewhat 
longer  than  the  fusiform  bacilli. 
Microorganisms,  as  staphylococci, 
streptococci  and  not  infrequently 
diphtheria  bacilli,  usually  accompany 
the  microorganisms  of  Vincent's 
angina,  and  by  reason  of  this  fact  it 
is  impossible  to  decide  that  the  fusi- 
form bacilli  and  spirilla  are  primary 
etioligical  factors.  Animal  inocula- 
tion with  these  microorganisms  has 
led  to  little  results. 

Various  fusiform  bacilli  which  morph- 
ologically are  indistinguishable  from 
those  found  in  Vincent's  angina  may 
frequently  be  found  from  smears 
from  the  gums,  from  carious  teeth, 
and  occasionally  mixed  with  micro- 
organisms in  the  pus  from  old  sinuses. 
Weaver  and  Tunnicliff  have  found 
spirilla  and  fusiform  bacilli  in  great 
numbers  present  in  a  gangrenous  dis- 
ease of  the  gums  and  cheeks,  called 
noma.  Here  again,  it  is  uncertain 
whether  the  organisms  are  primarily 
the  etiological  factor  in  the  disease 
or  merely  secondary  invaders. 

Spiroch.eta  Cbermeieri  (relapsing  fever). 
This  organism  was  discovered  by 
Ob«rmeier  in  1873  in  the  l)lood  of 
patients  suffering  from  relapsing 
fever.  The  organisms  are  long, 
slender,  flexible,  spiral  or  wavy  fila- 
ments with  pointed  ends,  with  from 


298  BACTERIOLOGY. 

four  to  ten  or  more  undulations. 
Compared  with  the  red  blood  cells 
among  which  they  are  seen,  the  or- 
ganiam  may  vary  from  one-half  to 
ten  times  the  diameter  of  a  corpuscle. 
They  stain  somewhat  faintly  with 
watery  solution  of  basic  aniline  dyes, 
and  stain  best  by  the  Romanowsky 
method  or  its  modification.  They  are 
negative  to  Gram.  A  terminal  flagel- 
lum  has  been  demonstrated  by  Novy. 

The  organisms  are  found  in  the  blood 
or  blood  organs  and  never  in  the 
secretions,  and  only  during  the  fever 
and  not  in  intermission.  In  the  fresh 
preparation  from  the  blood,  they  ex- 
hibit active  movements  accompanied 
by  very  rapid  rotation  in  the  long 
axis  of  the  spiral  filaments,  or  un- 
dulating movement.  Their  movements 
will  be  active  for  a  considerable  time 
if  kept  in  blood  serum  in  0.6  per  cent 
sodium  chloride  solution.  They  are 
killed  quickly  at  60°  C.  but  will  re- 
main alive  some  time  at  0°  C.  Many 
unsuccessful  attempts  at  cultivation 
have  been  made.  Novy  finally  suc- 
ceeded in  cultivating  them  in  celloidin 
capsules  placed  in  the  peritoneal  cav- 

*  ity  of  rats. 

The  disease  has  been  produced  in 
monkeys,  rats  and  mice  by  inoculat- 
ing them  with  the  blood  containing 
the  spirochetes.  In  man  the  organism 
produces  the  following  symptoms: 

The  microorganism  was  found  in  a 
large  percentage  of  the  cases  exam- 
ined, both  in  the  cutaneous  papules 
and  in  ulceration.  He  determined  that 
no  monkeys  are  susceptible  to  inocu- 
lation. The  monkeys  susceptible  to 
inoculation  with  yaws  do  not  become 
immune  to  syphilis,  neither  do  those 
having  syphilis  become   immune  for 

yaws.  Further  specific  differences  be- 
tween the  two  disease  have  been 
shown  by  the  Bordet-Gengou  reaction. 
By  reason  of  the  morphological  simil- 
arity to  the  tryponema  pallidum,  it 
should  probably  be  called  treponema 
pertenus. 

Spirocheta  GalUnamm  is  an  acute  in- 
fectious disease  occuring  among 
chickens,  chiefly  in  South  America. 
It  is  caused  by  a  spirocheta  which, 
morphologically,  is  very  similar  to 
the  spirocheta  of  Obermeirei.'    It  Is 


BACTERIOLOGY.  299 


easily  demonstrated  in  the  circulating 
blood  by  staining  the  blood  with 
Giemsa's  stain  or  by  dilute  carbo 
fuschin.  The  organism  has  lately 
been  successfully  cultivated  by 
Noguchi*  under  anaerobic  conditions. 
The  disease  has  been  transmitted 
from  animal  to  animal  by  subcu- 
taneous injection  of  blood.  Other 
birds  are  susceptible  as  well.  Mam- 
mals have  not  been  successfully  in- 
oculated. The  disease  is  generally 
transmitted  to  the  chicken  by  a  spe- 
cies of  tick  which  acts  as  an  inter- 
mediate host  and  causes  the  infection 
by  a  bite.  Active  immunization  may 
be  carried  out  by  the  injection  of  in- 
fected blood  in  which  the  spirochetes 
have  been  killed.  The  serum  of  im- 
mune animals  has  a  protective  action 
upon  birds.  Sacharoff  had  previously 
reported  an  organism  named  spi- 
rochete anserina,  which  caused  a  dis- 
ease in  geese,  principally  in  Russia 
and  Northern  Africa,  which  clinically 
and  pathologically  corresponds  to  the 
disease  caused  by  the  spirocheta  gal- 
linarum,  and  it  is  not  impossible  that 
these  two  organisms  may  be  identical. 

A  "rapid  rise  of  temperature  which  re- 
mains high  for  five  to  seven  days 
then  drops  by  crisis.  At  the  end  of 
seven  days,  another  rise  of  temper- 
ature is  followed  by  an  earlier  crisis. 
There  may  be  a  second  or  third  re- 
lapse. The  organisms  multiply  rapid- 
ly in  the  blood  from  the  beginning 
of  the  fever.  They  begin  to  disappear 
a  short  time  before  the  crisis,  and 
after  the  crisis  it  is  nearly  impossible 
to  find  them  in  the  circulating  blood. 
The  disease  is  not  often  fatal.  The 
mortality  in  different  epidemics  varies 
from  ten  to  two  per  cent. 

The  mode  of  transmission  of  the  disease 
is  not  clear,  though  infection  prob- 
ably occurs  through  the  bite  of  blood 
sucking  insects.  In  the  African  dis- 
ease the  transmission  occurs  through 
the  intermediation  of  a  tick  (Orni- 
thodoros  moubata),  which  infects 
itself  when  sucking  blood  from  an 
infected  human  being. 

Recovery  from  an  attack  usually  results 
in  a  more  or  less  definite  immunity. 
The  individuals  who  have  recovered 
have  hyper  immunized  blood.  Both 


300  BACTERIOLOGY. 


the  active  and  passive  immunity  may 
last  for  months. 
Spirocheta  Duttonl.  Button  in  1905 
showed  the  cause  for  African  tick 
fever  to  be  due  to  an  organism  that 
was  morphologically  very  similar  to 
the  SP.  Obermeieri.  Novy  and  Frankel 
believed  that  this  organism  is  another 
variety  if  not  another  species  of  the 
group. 

The  organism  can  be  transferred  to 
monkeys  by  the  bites  of  young  ticks 
at  their  first  feeding,  after  hatching 
from  infected  parents.  Button  acci- 
dentally inoculated  himself  through  a 
break  in  the  skin  while  performing  an 
autopsy  upon  an  infected  subject,  and 
died  from  the  disease. 

Spirocheta  Carter!  was  described  by 
Carter  in  1877  as  causing  relapsing 
fever  in  Bombay. 

Spirocheta  Pallida  (Treponema  Palli- 
dum). Schaudinn  working  with  Hoff- 
man, in  1905,  while  investigating  a 
number  of  primary  syphilitic  indura- 
tions and  secondary  large  lymphnodes, 
discovered  a  spirochete  and  named  it 
spirocheta  pallida.  He  thought  that 
the  organism  was  the  cause  of  the 
»  disease.  Further  study  by  him  re- 
vealed a  delicate  flagellum  at  each 
end,  but  left  the  existence  of  the 
undulating  membrane  which  he  at 
first  thought  present  in  doubt,  so  he 
called  it  tryponema  pallidum.  Ex- 
tensive studies  of  human  syphilis  and 
experimental  syphilis  of  lower  an- 
imals has  since  corroborated  the 
work  of  Schaudinn  and  Hoffman.  The 
organism  is  a  very  delicate  structure, 
closely  resembling  the  spirocheta 
dentium  in  morphology  and  staining 
reaction.  The  spirals  number  from 
four  to  twenty  and  are  quite  deep. 
The  angle  of  the  spiral  turn  is  very 
short.  There  are  anterior  and  flagella- 
like  prolongations.  On  rare  occasions 
double  flagella  appear  at  one  end, 
which  Schaudinn  interprets  as  be- 
ginning longitudinal  division.  Alive 
the  organism  is  not  very  refractive, 
hence  seen  with  difficulty.  Its  char- 
acteristic movements  are  rotation  on 
its  long  axis,  quivering  movem,ents  up 
and  down  the  spirals,  slight  forward 
and  backward  motion  and  bending  of 
the  entire  body.    The  organism  stains 


BACTERIOLOaY.  301 


red  by  Giemsa's  method,  as  does  also 
the  spirocheta  dentium.  Mo®t  other 
spirochetes  stain  blue.  The  organisms 
have  been  found  constantly  present  in 
the  primary  and  secondary  lesions  of 
all  carefully  investigated  cases.  The 
presence  of  the  spirochetes  in  the 
blood  has  been  demonstrated  by  van 
Bandi  and  Simanelli.  In  the  tertiary 
lesions  the  organism  is  found  less 
regularly  than  in  the  primary  and 
secondary  lesions.  In  congenital 
syphilis  the  organism  has  been  found 
in  the  lungs,  liver,  spleen,  pancreas, 
kidneys,  and  in  isolated  cased  in  the 
heart  muscle. 
The  organism  may  be  demonstrated  in 
the  living  state  by  the  hanging  drop 
method,  which  is,  however,  difficult 
and  uncertain.  A  better  method  is 
by  means  of  dark  field  illumination. 
The  material  taken  for  examination 
should  be  straight  from  syphilitic 
lesions,  and  if  not  dilute  enough  for 
examination,  it  should  be  emulsified 
in  a  drop  or  two  of  human  syphilitic 
fluid. 

The  organism  cannot  be  stained  with 
the  weaker  aniline  dyes,  therefore  the 
special  method  recommended  by 
Schaudinn  and  Hoffman  is  generally 
used. 

1.  Make  smears,  if  possible,  from  the 

depth  of  the  lesion  and  free  as 
possible  from  blood. 

2.  .Fix  in  methyl  alcohol  for  ten  to 

twenty  minutes  and  dry. 

3.  Cover  with  a  fresh  solution  of: 

Distilled  water  .10  cc. 

V  Potassium   carbonate,   1    to  1000 

 5  to  10  drops. 

Giemsa's   solution.  10  to  12  drops. 
Allowing   the   mixture   to   act  for 
one  to  four  hours,  preferably  in 
a  moist  chamber. 

4.  Wash  in  running  water. 

5.  Blof  and  examine. 

The    organism    is    stained    with  a 

violet  or  reddish  tint. 
The  organism  may  be  demonstrated 

in    tissues    by    the    method  of 

Levaditi. 

1.  Fresh    tissue    is    cut    into  small 

pieces  of  two  to  four  m.m.  thick- 
ness. 

2.  Fix  in  ten  per  cent  formula  for  24 

hours. 


302  BACTERIOLOGY. 

3.  Wash  in  water. 

4.  DeiJnydrate   in   96%  alcohol  for  24 

hours. 

5.  Wash  in  water. 

6.  Place  in  a  three  per  cent  silver  ni- 

trate solution  at  a  temperature  of 
37%°  C.  and  in  the  dark  for  three 
to  five  days. 

7.  Wash  in  water. 

8.  Place  in  a  freshly  prepared  solu- 

tion of: 

Pyrogallic  acid  2  to  4  g-rams 

Formalin  5  cc 

Distilled  water  100  cc. 

Allow  to  remain  in  this  solution  for 
24  to  48  hours  at  room  temper- 
ature. 

9.  Wash  in  water. 

10.  Dehydrate  in  graded  alcohol. 

11.  Embed   in  parafiine   and   cut  thin 

sections. 

12.  Examine  without  further  staining 

or  counter  stain  with  Giemsa's 
solution  or  Hemotoxin. 

Attempts  at  cultivation  were  at  first 
unsuccessful.  Later  cultivations  have 
been  reported  by  Schereschewsky  and 
Muehlens  but  did  not  succeed  in  car- 
rying out  Koch's  postulates  with  the 
c^iltures  they  obtained. 

Noguchi  has  successfully  cultivated  the 
spirochete  as  follows: 

Into  tubes  (20  cm.  high  and  1.5  cm. 
wide)  he  placed  10  cc.  of  a  serum 
water  made  of  three  parts  of  distilled 
water  and  one  part  of  coarse  sheep 
or  rabbit  serum.  These  were  steril- 
ized by  the  fractional  method,  after 
which  a  small  piece  of  sterile  rabbit 
kidney  or  testicle  and  a  bit  of  testicle 
of  syphilitic  rabbit  were  placed  in 
each  tube. 

The  serum  in  the  tubes  was  now  cov- 
ered with  sterile  parafl^in  oil  and 
placed  in  an  anaerobic  jar  at  33%°  C. 
for  ten  days,  at  which  time,  the 
spirocheta  had  developed  greatly  to- 
gether with  bacteria.  He  obtained 
pure  cultures  from  these  cultivations 
by  allowing  the  spirochetes  to  grow 
through  Berkefeld  filters;  also  by 
what  he  considers  a  better  method, 
preparing  high  tubes  of  three  parts  of 
very  slightly  alkaline  or  natural  agar 
to  which  a  piece  of  sterile  tissue  had 
been  added.  These  tubes  are  inocu- 
lated from  the  impure  cultures  with  a 


BACTERIOLOGY.  303 

long-  pipette.  The  spirocheta  and  bac- 
teria grow  close  to  the  tissue  and 
along-  the  stab.  At  the  end  of  ten 
days  to  two  weeks,  the  spirocheta 
wander  from  the  stab  and  appear  as 
hazy  colonies.  The  tubes  are  cut  and 
the  colonies  are  directly  transplanted 
to  other  serum  agar  tissue  tubes. 
Noguchi  carried  out  Koch's  postulates 
with  syphilis. 
So  far  as  is  known,  syphilis  in  nature 
appears  only  in  man.  All  experi- 
mental inoculations  of  animals  were 
unsuccessful  until  MetchnikoflC  and 
Roux  (1903)  succeeded  in  transmit- 
ting the  disease  to  a  female  chim- 
panzee. Klebs  stated,  1879,  that  he 
had  produced  syphilis  in  monkeys  by 
the  inoculation  of  human  virus.  Since 
then  Lazear  has  also  successfully  in- 
oculated monkeys.  Nicolle  succeeded 
in  inoculating  the  lower  monkeys 
(Macacus)  with  syphilis.  Attempts 
to  transmit  syphilis  from  the  tertiary 
lesions  have  been  unsuccessful.  The 
org^anisms  can  be  demonstrated  both 
in  primary  lesion  and  in  the  second- 
ary enlarged  gland  of  the  inoculated 
animal.  Bertarelli  produced  an  ulcer- 
ative lesion  of  syphilis  by  inoculating 
upon  the  cornea  and  into  the  anterior 
chamber  of  the  eye,  and  later  found 
the  spirocheta  within  this  situation. 
Syphilis  generally  remains  localized 
in  rabbits  as  well  as  in  the  lower 
monkeys.  Parodin,  in  1907,  inoculated 
syphilis  into  the  testicles  of  rabbits, 
and  this  method  has  proven  to  be  the 
most  simple  in  obtaining  the  spi- 
rocheta from  lesions  in  man  and  in^ 
definitely  carried  along  by  continuous 
transinoculation  from  one  rabbit  to 
another. 

After  the  development  of  the  primary 
lesion  man  is  usually  insusceptible 
to  reinoculation  during-  the  active 
stage  of  the  disease,  but  in  some  cases 
both  man  and  monkey  can  be  reinocu- 
lated.  Reinoculation  in  the  tertiary 
state  produced  precocious  lesions  of 
tertiary  type,  gumma  and  tubercles. 
Injections  of  large  quantities  of 
syphilitic  serum  into  chimpanzee  has 
failed  to  produce  definite  immunity. 

For  the  Bordet-Gengou  phenomena  see 
Wasserman  reaction  under  Comple- 
ment Fixation. 


304  BACTERIOLOGY. 


Spiroclieta  Pertenuis  (Framboesia  trop- 
ica, yaws).  Yaws,  a  disease  resem- 
bling- syphilis,  occurs  in  tropical 
and  subtropical  countries,  and  Castel- 
lan! in  1906  announced  that  he  had 
found  a  spiroorganism  which  bore  a 
close  resemblance  to  the  spirocheta 
pallida.  He  named  it  spirocheta 
pertenuis. 

Spiroolieta  Fhasredenls  is  an  organism 
of  probably  a  new  species  cultivated 
by  Noguchi  from  the  phagedemic 
lesions  on  human  external  genitals. 

Spiroclieta  Macro dentium  is  believed  by 
Nog-uchi  to  be  identical  with  Vincent's 
spirocheta. 

Spirocheta  Microdentium  cultivated  by 
Noguchi  from  the  tooth  deposits  in 
children. 

Spirocheta  Calligrymm  cultivated  by 
Noguchi  from  condylomata  is  prob- 
ably a  new  species. 


THE  BACTERIOLOGY  OF  I^IILK. 

The  use  of  cow's  milk  as  a  food,  espe- 
cially for  infants,  has  caused  it  to  be 
closely  studied. 

Milk  usually  contains  about  87%  of 
water  and  about  13%  of  solids.  Of  the 
solids  there  is  approximately  4%  of  fat; 
the  remaining-  9  7o  is  composed  of  about 
5%  lactose,  about  3.3%  protein  (casei- 
nogen  4  parts,  albumen  1  part)  and 
about  .7%  of  ash  (salts).  There  are  in 
addition  hydrolitic  enzymes  as  galac- 
tase,  a  proleolytic  enzyme  and  oxidase. 

Milk  is  a  favorable  culture  medium 
for  the  development  of  bacteria  and 
"therefore  very  well  fitted  to  convey  the 
germs  of  infectious  diseases.  - 

It  is  ordinarily  impracticable  to  se- 
cure milk  entirely  free  from  bacteria. 
In  the  milk  ducts  and  in  the  teats  of 
even  healthy  cows  a  certain  number  of 
bacteria  may  be  found,  although  within 
the  udder  milk  is  sterile. 

If  pyogenic  or  systemic  disease  of 
bacterial  origin  exists,  the  milk  may  be 
infected. 

Certain  forms  of  bacteria  seem  to  de- 
velop within  the  milk  cistern  and  within 
the  larger  milk  ducts.  The  first  milk 
drawn  from  the  teats  is  generally  loaded 
with  bacteria,  in  the  later  milk  they  are 
comparatively  few  in  number  in  com- 
parison. 


BACTERIOLOGY.  305 


Usually  milk  drawn  from  the  udder 
contains  less  than  100  bacteria  per  1 
C.C.,  although  in  some  cows,  seemingly 
normal,  there  may  be  large  numbers. 

There  are  changes  taking  place  In 
milk,  due  to  micro-organisms,  which  in 
a  sense  may  be  considered  normal  and 
may  be  divided  into  a  stage  of  bacterial 
action,  a  development  of  lactic  acid,  a 
neutralization  of  lactic  acid  and  a  de- 
composition or  putrefaction. 

Incidental  changes,  brought  about  by 
bacteria,  such  as  sweei  curdling,  ropy, 
soapy  or  color  formation  may  infre- 
quently be  met  with. 

The  most  important  source  of  bacteria 
in  milk  is  probaly  due  to  contamination 
of  the  milk  from  dust  particles  contain- 
ing bacteria,  which  are  dislodged  from 
the  hair  and  skin  of  the  udder  and  sides 
of  the  cow  during  the  process  of  milk- 
ing. It  is  therefore  necessary  to  have 
the  animal  carefully  groomed  and  adja- 
cent body  surfaces  thoroughly  moisten- 
ed in  order  that  this  source  of  contami- 
nation may  be  eliminated  while  milking. 
The  organisms  from  this  origin  are 
largely  of  fecal  origin. 

Dust  in  the  building  in  which  cows 
are  milked,. from  dusty  fodder  or  bedding, 
is  also  a  source  of  contamination,  and 
are  usually  the  B.  subtilis  and  putrefac- 
tive types  of  bacteria. 

The  hands  of  milkers,  unless  carefully 
cleaned,  will  also  afford  an  opportunity 
for  milk  infection.  This  contamination 
may  more  readily  carry  infection  from 
the  milkers  to  other  individuals,  than  an 
infection  from  the  cow  itself  to  man. 

Milking  utensils  may  also  prove  a 
dangerous  source  of  infection,  in  that 
imperfectly  soldered  joints  may  harbor 
innumerable  bacteria.  Utensils  should 
be  thoroughly  scalded,  or  the  entire  ves- 
sel heated  to  the  boiling  point  of  water 
to  destroy  the  organisms  present. 

Careless  handling,  such  as  allowing 
milk  to  stand  in  open  cans  or  the  use  of 
unclean  dippers,  etc.,  or  contaminated 
water  used  for  rinsing  milk  vessels,  is 
frequently  a  cause  of  contaminating 
clean  milk. 

The  number  of  bacteria  in  fresh  milk 
will  decrease  for  a  time,  which  indicates 
a  germicidal  action.    The  length  of  time 


306  BACTERIOLOGY. 

of  bactericidal  action  differs  with  the 
number  and  kinds  of  bacteria  and  with 
the  conditions  under  which  the  milk  is 
kept.  Arguments  as  to  the  reduction  of 
bacteria  are  many.  Some  hold  that  re- 
duction is  due  to  agglutinating  power  of 
milk,  so  that  in  reality  there  is  no 
actual  reduction  in  bacterial  number. 
Others  argue  that  all  bacteria  gaining 
entrance  to  milk  do  not  find  favorable 
environment  and  die  more  rapidly  than 
those  which  find  environment  suitable. 

It  would  seem,  however,  that  milk 
must  contain  a  certain  amount  of  bac- 
tericidal action  by  reason  of  germicidal 
substances  contained  in  blood,  which 
must  be  given  off.  at  least  in  part,  with 
the  milk. 

The  number  of  leucocytes  present  in 
milk  would  also  be  a  factor,  as  their 
phagocytic  power  would  not  be  lost  im- 
mediately with  the  milking. 

The  bactericidal  property,  however, 
can  in  no  case  completely  sterilize  milk, 
as  the  bactericidal  action  is  specific, 
that  is,  certain  bacteria  are  destroyed 
by  it,  while  others  are  not  affected. 

The  lactic  acid  organisms,  present  in 
m'ilk  develop  rapidly,  particularly  if 
milk  is  kept  in  a  warm  place.  When  these 
organisms  develop  0.4%  of  lactic  acid 
the  milk  will  be  decidedly  sour  in  taste. 
When  .75%  to  .80%  acidity  is  reached, 
curdling  of  milk  takes  place. 

The  ordinary  lactic  acid  bacteria  will 
rarely  produce  more  than  a  1.25%  acid- 
ity. The  Bact.  bulgaricum  group  of 
bacteria  will,  however,  produce  a  much 
higher  percentage  of  acidity. 

Sour  milk  may  be  kept  under  anaero- 
bic conditions  for  a  long  time  without 
producing  any  change  in  its  composition. 
If  exposed  to  the  air,  however,  certain 
mords  (e.g..  Oidium  lactis)  develop  on 
the  surface* of  the  milk,  using  the  lactic 
acid  as  food,  oxidizing  it  to  CO2  and 
water.  By  reason  of  this  the  acidity  of 
the  milk  is  neutralized.  Some  of  the 
acid  may  also  be  neutralized  by  the  milk 
caseinogen. 

When  the  excess  milk  acidity  has  been 
neutralized,  the  various  putrefactive 
bacteria  develop  and  the  milk,  particu- 
larly the  caseinogen,  rapidly  decom- 
poses. 


BACTERIOLOGY.  307 


Milk  heavily  inoculated  with  the  B. 
subtilis  group  of  organisms  may  not 
sour,  but  undergo  sweet  curdling  in- 
stead. This  is  due  to  the  overgrowth  of 
the  lactic  acid  organisms  and  the  pro- 
duction of  a  rennet-like  enzyme  by  the 
subtilis  group.  The  curd  is  later  more 
or  less  completely  digested. 

Certain  organisms  produce  the  so- 
called  ropy  milk  by  the  formation  of 
gums  from  carbohydrates  and  mucin- 
like  substances  from  the  proteins,  while 
certain  other  organisms  may  produce 
red,  yellow,  blue  and  even  black  milk. 

The  undesirable  flavors,  sometimes 
produced  in  milk^  characterized  as  soapy 
and  bitter  milks,  are  produced  by 
bacteria. 

The  number  of  bacteria  present  in  a 
given  sample  of  milk  depends  upon  the 
contamination  taking  place  during  the 
milking  process,  the  time  which  elapses 
after  the  milking,  the  temperature  at 
which  the  milk  is  held,  the  care  in 
handling  and  the  matter  of  non-pasteur- 
ization or  pasteurization. 

The  temperature  at  which  milk  Is 
held  is  very  important.  The  acid-pro- 
ducing organisms  and  most  other  forms 
grow  slowly  if  at  all  at  low  tempera- 
tures. Milk  should  therefore  be  cooled 
as  soon  as  possible  after  drawing  and 
kept  at  a  low  temperature  so  as  to  pre- 
vent the  multiplication  of  bacteria.  If 
this  is  carried  out  milk  may  be  kept 
from  souring  several  days. 

If  it  is  not  quickly  cooled  and  is  kept 
at  room  temperature  it  may  sour  In  less 
than  24  hours. 

The  number  of  bacteria  may  be  great- 
ly reduced  by  pasteurization. 

Infectious  transmitted  "by  mlllc. 

The  most  important  infections  trans- 
mitted by  milk  are  the  diarrhoeas  and 
dysenteries  of  infants.  The  intestinal 
tracts  of  infants  seem  particularly  sus- 
ceptible to  infection  of  micro-oranisms 
belonging  to  the  enteriditis,  paratyphoid 
and  dysentery  groups.  The  summer 
complaints  of  infants  are,  in  large  part, 
due  to  the  use  of  milk  containing  these 
organisms.  Wherever  it  is  impossible  to 
obtain  an  infection-free  milk  for  infant 
feeding,  pasteurization  becomes  neces- 
sary. 


308  BACTERIOLOGY. 


Typhoid  fever  epidemics  have  fre- 
quently been  traced  to  an  infection 
through  the  milk  supply.  This  is  also 
true  for  scarlet  fever  and  diphtheria. 

The  use  of  tuberculous  milk  is  the 
common  cause  of  a  number  of  cases  of 
tuberculosis  in  children,  the  milk  hav- 
ing been  contaminated  by  the  organism 
entering  the  milk  within  the  udders  of 
cows  or,  what  is  more  likely,  by  con- 
tamination through  the  feces  of  animals 
sick  with  the  disease. 

Milk  should  come  from  herds  that 
have  been  tested  by  tuberculin,  and  from 
which  all  of  the  tuberculous  animals 
have  ^  been  removed. 

Anthrax,  foot  and  mouth  disease,  and 
malta    fever    have    infrequently  been 
transmitted  through  milk. 
Milk  Analyses. 

1.  Plate  various  dilutions  of  milk  on 
nutrient  agar  (+10  reaction). 

2.  Incubate  at  37°  C.  for  48  hours,  or 
at  22°  C.  for  5  days. 

Milk  properly  drawn  will  not  contain 
more  than  500  to  1000  bacteria  per  1  c.c. 
Milk  as  sold  in  cities  is  from  36  to  48 
hours  or  over  old  before  use,  contains 
many  times  the  above  number. 

Good  milk  may  contain  about  1,000,000 
bacteria  per  1  c.c,  or  even  more  when  it 
begins  to  sour,  so  that  it  is  evident  that 
numbers  alone  are  of  little  moment  ex- 
cept that  they  indicate  the  care  used  in 
milking  and  delivery  to  the  consumers. 

Certain  cities  have  classified  milk  into 
uninspected  milk,  inspected  milk,  pas- 
teurized milk  and  certified  milk. 

Uninspected  milk  has  no  sanitary  con- 
trol. 

Inspected  milk  is  a  milk  which  comes 
from  cows  tuberculin  tested,  and 
which  is  drawn  and  cared  for  under 
sanitary  conditions. 

Pasteurized  milk  is  a  milk  which  has 
been  heated  for  a  short  period  of 
time  at  a  temperature  considerably 
below  the  boiling  point,  and  then 
followed  by  a  rapid  chilling.  Its 
object  is  the  destruction  of  harmful 
bacteria  and  their  products. 

The  two  methods  of  pasteurization 
are: 

(1)  The  "holder  process"  in  which 
the  milk  is  heated  to  60-65°  C.  and 


BACTERIOLOGY.  309 


held  at  this  temperature  for  about 
one-half  hour.     It  is  then  cooled 
rapidly  and  bottled. 
(2)  The  "flash  or  continuous  pro- 
cess" in  which  the  milk  is  heated 
to  80-85°  C.  and  held  at  this  tem- 
perature  for    30    seconds    to  one 
minute.     It    is    then    cooled  and 
kept  at  a  low  temperature  until 
distributed. 
The  holder  method  is  held  to  be  the 
most  efficient,  as  it  destroys  tiie  larger 
percentage  of  bacteria. 

Certified  milk  is  now  a  milk  obtained 
from  animals  free  from  contagious 
or  infectious  disease;  ^ attendants 
must  be  in  good  health;  stables 
must  be  sanitary,  well  lighted,  and 
free  from  dust;  milking  vessels 
must  be  sterile,  and  every  precau- 
tion must  be  used  to  prevent  the  en- 
trance of  bacteria  to  the  milk.  After 
milking  it  must  be  quickly  cooled, 
sealed  in  bottles,  and  kept  cold  until 
delivery.  In  most  cities  where  cer- 
tified milk  is  inspected  it  must  not 
contain  more  than  10,000  bacteria 
per  1  c.c. 

THE  BACTERIOLOGY  OF  WATER. 

All  natural  waters  contain  micro-or- 
ganisms, which  gain  entrance  from 
many  sources.  The  vapors  arising  from 
the  sea  or  land  contain  no  organisms, 
but  as  soon  as  precipitation  takes  place, 
the  organisms  enter  the  water  from  the 
air  and  soil. 

Certain  organisms,  because  of  their 
ability  to  find  sufficient  nutriment  for 
life  and  growth  in  water,  may  be 
spoken  of  as  belonging  to  the  "water 
flora."  Some  bacteria,  found  in  water, 
flourish  only  during  rain  and  flood  sea- 
sons, while  other  bacteria,  such  as  in- 
testinal organisms  survive  for  a  short 
period  only. 

The  organisms  found  in  water  may  be 
divided  into: 

1.  The  Natural  Water  Bacteritt,  which 
are  frequently  numerous  and  gener- 
ally harmless  to  man.  Certain  species 
will  predominate  at  one  season  and 
disappear  at  another.  Some  bacteria 
contained  in  water  have,  by  reason  of 
their  biochemical  properties,  been  di- 
vided into: 


810  BACTERIOLOGY. 


(a)  A  bacillus  fluorescence  llquefa- 
ciens  group,  recognized  by  the  green 
fluorescence  of  the  colonies  and  liq- 
uefaction of  gelatine,  is  more  fre- 
quently found  in  water  than  in  any 
other  form. 

(b)  A  bacillus  fluorescence  non-llque- 
faciens  group,  produce  the  charac- 
teristic fluorescence,  but  do  not 
liquefy  gelatine,  are  very  abundant 
in  river  water  "and  are  represented 
by  the  B.  f .  longus,  B.  f .  tennis,  B.  f. 
aureus  and  B.  f.  crassus. 

(c)  A  groxLj?  of  liquefy ing-  and  milk 
acidifying  bacilli.  These  are  com- 
n^on  to  certain  seasons.  Some  are 
soil  organisms,  some  are  related  to 

,  the  proteus  group,  others  are  the  B. 
liquefaciens,  B.  punctatus  and  B. 
circulans. 

(d)  A  chromog'enic  bacilli  group  such 
as  B.  prodigiosus,  B.  ruber,  B.  indi- 
cus  B.  rubescens,  B.  rubefaciens,  B. 
aquatilis,  B.  ochracens,  B.  aurantia- 
cus,  B.  fulous,  etc.,  are  often  pres- 
ent in  water. 

At  certain  times,  in  river  and  brook 
waters,  violet  organisms  as  B.  violacius 
or  B.  janthinus,  B.  lividus,  B,  amie- 
thy'stinus  and  B.  coerulens  are  found. 

(e)  A  chromogenic  cocci  group.  Sar- 
cina  lutea  is  the  most  common  spe- 
cies, though  this  group  is  not  nu- 
merous in  water. 

(f)  A  non-chromog'enic  cocci  group, 
such  as  the  non-liquefying  M.  can- 
dicans,  M.  nivalis,  M.  aquatilis,  and 
a  liquefying'  type  as  the  M.  coro- 
natus. 

2.    Soil    bacteria    from  surface  wash- 
ing-.    Numerous  soil  organisms  are 
found    in    natural    waters  during 
floods  and  after  rains.    Certain  spe- 
cies of  these  organisms  may  remain 
in  the  water  for  a  long  time. 
The  most  common  organisms  of  this 
group  are  the  B.  mycoides,  B.  subtilis, 
B.  megatherium,  B.  mesentericus  (vul- 
gatus,  fuscus  and  ruber). 

The  colonies  of  these  organisms  are 
characteristic  rhizoid,  liquefying  -gela- 
tine, and  produce  spores.  One  of  the 
thread  bacteria  (cladothrix  dichotoma) 
may  also  be  present.  It  is  frequently 
found  in  both  fresh  and  stagnant  water 
and  in  most  soils. 


BACTERIOLOGY.  311 


For  the  isolation  of  these  organisms, 
beef  peptone  gelatine  is  used.  "When 
other  media  are  used  a  different  flora, 
such  as  the  nitrofying  organisms,  yellow 
chromogens,  etc.,  appear. 

3.    Intestinal  Bacteria,  usually  of  sew- 
agre  origin. 

(a)  Protein  group.  The  B.  vulgaris, 
B.  zenkeri,  B.  mirabilis,  B.  zopfii,  B. 

cloacae  and  the  sewage  proteins  of 
Houston. 

These  organisms  are  very  abundant  in 
sewage,  but  are  not  present  in  very  large 
numbers  in  contaminated  water.  This 
group  of  organisms  are  mobile,  liquefy 
gelatine,  produce  gas  in  dextrose  and 
saccharose  broth  (sometimes  a  little  in 
lactose),  reduce  nitrates,  coagulate 
milk,  produce  indol  and  impart  a  fecal 
odor  to  the  media. 

(b)  Sewag'e  streptococci.  The  strepto- 
cocci in  water  is  indicative  of  re- 
cent sewage  contamination.  They 
die  quickly  outside  of  the  body.  By 
their  action  on  the  various  sugars 
an  equine,  human  and  bovine  type 
may  be  differentiated,  which  may  be 
used  as  indicative  of  recent  contam- 
ination from  street  washings,  hu- 
man excreta  or  cultivated  fields. 

(c)  B.  enteritidis  sporog'enes,  though 
usually  present  in  the  intestinal 
tract  of  man,  cannot  be  considered 
as  an  indicator  of  excretal  pollution 
by  reason  of  its  presence  in  dust, 
food  stuffs,  etc.,  and  the  resistance 
of  its  spores. 

(d)  B.  Coli.  The  bacillus  coli  is  ac- 
cepted as  the  bacterial  indicator  of 
sewage  pollution  of  water. 

(e)  Bact.  lactis  serog'enes,  next  to  B. 
coli,  may  be  regarded  as  an  indicator 
of  sewage  pollution  of  water. 

(f )  B,i  typhosus,  reported  to  have  been 
isolated  from  water  in  a  very  few 
instances,  will  live  in  pure  water 
from  8  to  10  days.  When  exposed  to 
the  action  of  sewage  bacteria,  it  will 
live  for  from  5  to  6  days. 

(g)  Msp.  comma.  The  spirillum  of 
Asiatic  cholera  is  an  intestinal  or- 
ganism and  spreads  the  disease 
largely  through  water.  It  has  been 
frequently  isolated  from  infected 
waters. 


312  BACTERIOLOGY. 


The  number  of  "bacteria  in  water. 

The  bacterial  purity  of  natural  waters 
depends  upon  the  source  from  which  the 
waters  are  derived,  together  with  the 
special  and  local  condition  in  relation  to 
contamination. 

Bain.  The  number  of  bacteria  present 
in  rain  water  depends  upon  the 
month  of  the  year  and  the  dryness 
of  the  air.  When  there  is  consider- 
able dust  in  the  air,  the  first  rain 
will  be  very  rich  in  bacteria,  but 
during  the  latter  hours  of  prolonged 
rain  the  water  may  be  comparative- 
ly sterile. 

The  rain  in  densely  inhabited  cities 
always  contains  more  bacteria  (av- 
eraging about  19  per  cc.)  than  the 
rain  falling  on  open  farm  land  or 
upland  pastures,  in  which  the  num- 
ber of  bacteria  will  average  about 
4.3  per  cc. 
Snow.  The  results  from  snow  fall  are 
similar  to  those  from  rain,  except 
that  the  number  of  bacteria  present 
per  cc.  is  larger;  334  to  463  bacteria 
per  1  cc.  of  snow  water  has  been 
recorded,  while  Binot  did  not  find  a 
•single  mocro-organism  present  in  8 
cc.  of  water  from  mountain-top 
snow. 

Hail  Stones.  The  number  of  bacteria 
obtained  from  hail  stones  varies 
from  628  to  21,000  per  cc.  by  reason 
of  surface  water  being  carried  by 
storms. 

Well  Water.  Deep  well  water  ordinarily 
contains  but  few  organisms.  Usu- 
ally less  than  50  per  cc.  on  gelatin 
at  20°  C.  and  about  5  per  cc.  on  agar 
plates. 

Shallow  well  water's  bacterial  content 
varies  with  the  amount  of  rain  fall, 
even  though  they  are  well  located 
and  constructed.  The  water  in  pol- 
luted wells  may  contain  enormous 
numbers  of  organisms;  20,000  bac- 
teria per  cc.  on  gelatin  has  been 
reported. 

Spring"  Water  corresponds  in  bacterial 
content  to  that  of  deep  wells. 

Upland  Surface  Water  contains  but  few 
bacteria  if  draining  from  barren 
land.  Cultivation  and  habitation 
may  change  this  considerably. 


BACTERIOLOGY.  313 


Pure  waters  contain  from  50  to  300 
bacteria  per  1  cc.  when  grown  on 
g-elatin  and  from  1  to  10  on  agar. 
River  Water.  The  bacterial  content  of 
river  water  is  influenced  by  sewage 
contamination  temperature,  rain 
fall,  vegetable  debris,  etc. 
Iiake  Water  is  generally  much  purer 
than  the  waters  of  rivers.  The  bac- 
terial content  near  the  shore  is 
greater  than  further  out  by  reason 
of  the  influence  of  habitation. 
Sea  Water  near  the  shore  and  in  the 
neighborhood  of  seaports  may  con- 
^  tain  a  large  number  of  bacteria.  In 
the  water  remote  from  the  coast 
there  are  few  bacteria. 
The  number  of  bacteria  in  natural 
waters  is  influenced  by: 
1.  Temperature.  A  low  temperature 
decreases  the  parasitic  types,  but 
the  number  of  other  bacteria  pres- 
ent during  the  hot  summer  months 
is  generally  somewhat  less  than 
during  the  cooler  months. 
Water  should  be  examined  for  its 
bacterial  content  immediately  after 
collection,  as  there  is  usually  a  re- 
duction in  the  number  during  the 
first  few  hours,  to  be  followed  later 
by  a  large  increase. 
The  samples  collected  for  analysis 
should  be  kept  cool,  although  very 
polluted  waters  snow  a  marked  de- 
crease of  intestinal  types  if  the 
sample  is  kept  cool. 
Iiigrht,  although  germicidal,  does  not  in- 
fluence the  number  of  bacteria  in 
water,  probably  "Dy  reason  of  the 
water's  turbidity  and  the  speed  of 
the  current.  The  greatest  germi- 
cidal effect  of  sunlight  is  produced 
in  shallow,  clear  and  slowly  moving 
water.  Direct  light  is  not  efficient 
as  a  purifier  of  water.- 
Pood  Supply.  In  water  containing  a 
large  amount  of  organic  matter  the 
number  of  bacteria  is  greatly  in  ex- 
cess of  that  in  which  there  is  but 
little  of  such  material.  Sewage 
water  is  rich  in  organic  matter  and 
therefore  contains  great  numbers  of 
bacteria.  The  number  of  bacteria 
present  in  a  given  water  is  therefore 
proportionate  to  the  diminution  of 
organic  material.    Self  purification 


814  BACTERIOLOGY. 

of  streams  is  dependent  mainly 
upon  the  causes  producing-  insuffi- 
ciency or  unsuitability  of  the  bac- 
teria's food  supply. 

Oxidation.  The  oxygen  absorbed  on  the 
surface  of  waters,  in  rapids,  falls 
and  tidal  rivers  can  be  considered 
as  a  very  minor  agent  in  the  purifi- 
cation of  water. 

Iiow  Plants  and  Animals  as  algse,  river 
plants  and  numerous  protozoa  re- 
duce the  organic  matter  of  water 
and  thereby  reduce  the  food  supply 
of  bacteria.  The  chemical  products 
of  hig-her  forms  are  injurious  to 
bacteria  and  many  bacteria  are  in- 
gested by  the  unicellular  animals. 

Dilution.  Polluted  water  flowing  into 
larger  bodies  of  pure  water,  as  into 
a  river  or  lake,  is  at  once  diluted, 
thus  diminishing  the  bacteria's  food 
supply,  likewise  also  diffusing  the 
bacteria  through  a  greater  volume 
of  water;  the  greater  the  dilution 
the  fewer  sewage  bacteria  will  .be 
found. 

Sedimentation.  .The  suspended  matter 
of  still  water  tends  to  sediment,  and 
this  in  itself  brings  about  its  puri- 
'fication. 

Water  Analyses. 

The  improbability  of  getting  typhoid 
bacilli  from  suspected  water,  except 
under  unusually  favorable  condi- 
tions, caused  a  return  to  the  esti- 
mation of  the  number  of  intestinal 
bacteria. 

It  is  known  that  the  group  of  colon 
bacilli  have  a  somewhat  longer  exist- 
ence than  the  typhoid  bacilli,  and  as  the 
colon  bacilli  come  chiefiy  or  wholly  from 
the  intestinal  passages  of  men  and  ani- 
mals, it  is  fair  to  assume  that  typhoid 
bacilli  could  not  occur  without  the  pres- 
ence of  the  colon  bacillus,  except  in 
rare  cases,  as,  for  example,  pollution 
with  urine  only.  The  latter  could  of 
course  occur  abundantly  without  the  ty- 
phoid bacillus. 

During  the  past  few  years  the  atten- 
tion of  sanitarians  has  been  seriously 
devoted  to  the  interpretation  of  the 
presence  of  smaller  or  larger  numbers 
of  colon  bacilli  in  water,  until  at  pres- 
ent upon  the  quantitative  analyses 
(measuring   within   certain   limits,  de- 


BACTERIOLOGY.  315 

composing  org-anic  matter)  and  the 
colon  test  (indicating  more  specifically 
that  pollution  derived  from  intestinal 
discharges  of  man  or  animals)  the  bac- 
teriological analyses  of  water  is  based. 

The  determination  of  the  number  of 
bacteria  is  also  of  value. 

TechxiicLue.  Utmost  care  is  necessary 
to  get  reliable  results.  A  speck  of 
dust,  a  contaminated  dish,  a  delay 
of  a  few  hours,  an  improperly  ster- 
ilized agar  or  gelatin,  a  too  high  or 
too  low  temperature,  may  introduce 
an  error  or  variation  in  results 
which  would  make  a  reliable  test 
impossible. 
In  the  collection  of  sample: — 

1.  Utmost  carefulness  in  collection  is 
necessary. 

2.  An  immediate  test  is  essential  as 
bacteria  readily  increase  or  de- 
crease in  number  after  collection. 

Prankland  records  a  case  (well  water 
sample)  of  water  sample,  kept  for  3 
days  at  a  moderate  temperature,  in 
which  the  bacteria  increased  from  7  to 
495,000. 

Jordan  reports  a  case  of  sample  water 
in  which  the  bacteria  decreased  in  4'8 
hours  from  535,000  to  54,000. 

Park  and  Williams,  of  New  York  City, 
record  a  case  in  a  sample  from  Croton 
river  irt*^"  which  B.  Colon  present  in- 
creased from  10  to  100  per  c.c,  during 
24  hours. 

,  3.  It  is  better  to  make  cultures  in  the 
open  field  or  in  a  house,  rather 
than  to  wait  12  hours  for  the  con- 
veniences and  advantage  of  a  lab- 
oratory. 

4.  If  sent  to  the  laboratory,  water 
should  be  kept  at  about  5°C. 
(41°  P.)  during  transit. 

Quantity  of  water  to  "be  used  in  tests. 

1.  It  is  of  great  importance  to  add 
proper  amounts  of  water  to  the 
broth  in  the  fermentation  tubes 
and  in  the  media  for  planting. 
Usually  1  c.c.  and  0.01  c.c.  are 
added  to  the  fermentation  tubes 
and  10  c.c,  of  melted  nutrient  agar 
or  gelatin.' 

2.  If  possible  always  make  duplicate 
tests. 

3.  When  necessary  to  know  whether 
colon  is  present  in  larger  amounts 
than  1  c.c,  quantities  as  large  as 


316  BACTERIOLOGY. 


10  c.c.  or  100  c.c.  can  be  added  to 
bouillon,  and  then  after  a  few 
hours  1  c.c.  are  added  to  fermen- 
tation tubes. 

4.  Less  than  20  colonies  and  more 
than  200  on  a  plate  give  inaccurate 
counts,  the  smaller  number  being 
too  few  to  judge  an  average  and 
the  larger  number  interfere  with 
each  others  growth. 

When  as  many  as  10,000  colonies 
develop  in  the  agar  contained  in  one 
plate,  it  will  be  found  that  there 
will  develop  in  a  second  plate  con- 
taining but  1-10  the  amount  of 
water  from  20  to  50%  as  many 
colonies.  This  shows  that  crowd- 
ing of  the  colonies  had  prevented 
the  growth  of  all  but  1-5  to  %  of 
them. 

5.  The  chemical  composition  of  the 
medium  affects  the  results  of  the 
analyses. 

Nutrient  agar  of  a  1.5%  acid  re- 
action gives  slightly  lower  counts 
than  gelatin,  but  on  account  of  its 
convenience  in  summer  and  its 
greater  uniformity,  it  is  more  gen- 
erally used  for  routine  work. 
6..  The  American  Public  Health  Asso- 
ciation has  adopted  a  standard  re- 
action of  1%  acidity  which  is  the 
average  optimum  for  water  bacte- 
ria. 

Only  a  certain  proportion  of  bac- 
teria develop  and  all  we  can  ask  is 
that  our  count  represents  fairly* 
the  quick  growing  sewage  forms. 
7.  The  temperature  is  very  important. 
Plate  cultures,  as  a  rule,  are  grown 
at  20°C.-21°C.  for  days,  and  at  in- 
cubator temperature  (37.5°C.)  for 
from  24  to  48  hours. 
Some  bacteria  do  not  develop  in  4 
days,  but  these  are  neglected. 

The  number  ' of  bacteria  growing 
at  room  temperature  is  usually 
much  greater  than  those  growing 
at  37.5°C. 

As  all  the  intestinal  groups  of 
bacteria  grow  at  body  temperature, 
while  many  of  the  water  types  do 
not,  some  investigators  believe  it 
important  to  develop  the  bacteria 
at  both  temperatures  so  as  to  com- 
pare the  results.  (Advantage  in 
coli  tests). 


BACTERIOLOGY.  317 


8.  In  making  litmus  lactose  agar 
plates,  the  colon,  if  present,  will 
take  on  a  red  color  in  the  blue 
field.  If  many  coli  are  present 
the  whole  medium  becomes  red  by 
reason  of  the  acidity.  Later,  at 
48  hours  or  so,  by  reason  of  an 
alkali  being  produced  by  the  form- 
ation of  NH3,  the  blue  color  may 
return. 

Sigrnlficance  of  Coli  Bacilli  in  Water. 

The  colon  test  has  been  applied  with 
satisfaction  and  confidence  in  the  exam- 
ination of  water,  shell  fish,  and  other 
articles  of  food  by  many  authorities, 
while  other  authorities  have  denied  its 
value. 

Bacteriologists  have  found  bacilli  re- 
sembling certain  members  of  the  colon 
group  in  apparently  unpolluted  well 
water. 

The  discovery  that  animals  have  colon 
bacilli  identical,  in  the  usual  character- 
istics studied,  with  those  of  man  has 
complicated  matters. 

A  fresh  hill  side  stream  may  be 
loaded  with  colon  bacilli  from  the  wash- 
ings of  horse  or  cow  manure  used  as 
fertilizer  in  the  soil  of  the  field  through 
which  the  stream  runs,  or  the  stream 
may  be  polluted  by  a  stray  cow  or  horse. 
Swine,  hens,  birds  etc.,  may  contaminate 
in  unsuspected  ways. 

The  number  of  colon  bacilli,  rather 
than  their  presence,  in  any  body  of  sur- 
face water  is  therefore  of  importance. 

In  well  and  spring  water  the  presence 
of  the  colon  bacilli  indicates  contami- 
nation: The  absence  of  the  colon  bacil- 
lus in  water  proves  it  harmless  so  far 
as  bacteriology  can  prove  it. 

When  the  colon  is  present  in  numbers 
that  may  enable  one  to  isolate  it  from 
1  c.c.  quantities  in  a  series  of  tests, 
it  is  reasonable  proof  of  animal  or  hu- 
man contamination  ana  the  conditions 
should  be  investigated.  10  colon  in  1  c.c. 
indicates  serious  contamination. 

Surface  water  from  inhabited  regions 
will  always  contain  numerous  colon 
bacilli  after  a  heavy  rainstorm  or 
shower. 

The  washing  from  roads  and  culti- 
vated fields  contain  necessarily  large 
numbers  of  colon  bacilli. 

Wilson  reports  that  in  only  two  out 
of  58  samples  of  presumably  non-pol- 


318  BACTERIOLOGY. 


lute  waters  did  he  recover  colon  bacilli 
in  1  c.c.  samples;  even  in  21  stag-nant 
pools  he  only  found  colon  bacilli  in  5 
of  the  Ic.c.  samples. 

The  experience  of  all  those  who  have 
studied  the  subject  practically,  is  that  in 
delicacy  the  colon  test  surpasses  chemi- 
cal analysis;  in  constancy  and  definite- 
ness  it  also  excells  the  quantitative  bac- 
terial count.  All  tests  must,  however, 
be  supplemented  by  inspection. 

Analyses 

1st  Method. 

1.  Plate  1  c.c.  of  water  in  each  of  2 
or  3  Petri  dishes  containing  Hess's 
agar.  Incubate  for  from  24  to  48 
hours  at  37.5°c.,  at  end  of  which 
time,  count  the  colonies  appearing 
on  the  plate. 

2.  Plate  1  c.c.  of  water  in  each  of  2 
or  3  Petri  dishes  containing  gel- 
atin. Incubate  for  from  48  to  96 
hours  at  20-21°c.,  at  end  of  which 
time,  count  the  colonies  appearing 
on  the  plate;  make  note  of  the 
number  of  liquefiers. 

3.  Place  1  c.c.  of  water  in  each  of 
10  fermentation  tubes  containing 
dextrose  bouillon.  Incubate  at 
37.5''c.    for    48    nours.     Note  the 

»  quantity  of  gas  produced,  if  any, 
at' the  end  of  24  hours,  also  at  the 
end  of  48  hours.  Determine  the 
ratio  of  CO2  to  H. 

Fermentation    with    the  proper 
gas  ratio  in  but  one  tube  would 
suggest   that   1   colon   bacillus  is 
present  in  10  c.c.  of  water,  etc. 
Make  report  on  number  of  bacteria 

present  in  1  c.c.  of  water  grown  on 

agar. 

Make  report  on  number  of  bacteria 
present  in  1  c.c.  of  water  grown  on 
agar. 

Make  report  on  number  of  bacteria 
present  in  l.c.c.  of  water  grown  on 
gelatin. 

Make  report  on  number  of  liquefiers 
present  in  1  c.c.  of  water  grown  on 
gelatiu. 

Make  report  on  number  of  colon  bacilli 
present  in  1  c.c.  of  water  grown  in 
dextrose  bouillon. 
2iid  Method. 
1.   Plate  1  CO.  of  water  in  each  of  2 
or  3  Petri  dishes  containing  litmus 
lactose  agar  and  Incubate  at  37.5  "c. 


BACTERIOLOGY.  319 

for  from  24  to  48  hours,  at  end  of 
which  time  note  number  of  red 
colonies,  and  trasfer  these  to  each 
of  the  necessary  number  of  fermen- 
tation tubes  containing  1%  dex- 
trose bouillon.  Incubate  the  tubes 
for  24  hours.  If  gas  is  not  pres- 
ent, the  red  colonies  are  not  colon 
toacUli. 

If  g"as  is  present,  test  gas  ratio,  then 
apply  Riva's  test  for  colon  1,  2 
and  3. 

Blva  Test  No.  1. 

Boil  in  a  test-tube  about   5   c.c.  of 
media  from  the  fermentation  tubes, 
with  about  3  c.c.  of  a  10%  solution 
of  Na  OH. 
No  change  in  color=colon  bacillus. 
Change  in  color  to  a  pink=not  colon, 

but  ordinary  saccharolyte. 
Biva  Test  No.  2. 

Depends  upon  the  ability  of  colon  to 
exhaust  all  sugar  in  a  1%  dextrose 
bullion  in  24  hours.    Sugar  change 
as  a  matter  of  fact  ceases  at  the 
end  of  the  18th  hour. 
Determine   the    presence    or  absence 
of  sugar  in  the  media  of  fermentation 
tube  culture  by  boiling  a  small  quantity 
in  a  test-tube  containing  about  5  c.c.  of 
Pehling's  solution.    A  reduction  of  the 
copper  by  the  sugar  present,  changing 
blue  color  to  deep  yellow  to  red  ppt., 
indicates  colon  Ibacillus. 

No  reduction  of  the  copper,  if  no  sug- 
ar is  present,  consequently  no  change 
in  blue  color=:not  colon. 
Biva  Test  No.  3. 

Add  to  about  5  c.c.  of  fermentation 
tube  culture,  contained  in  a  test- 
tube*,  about  3  c.c.  of  a  50%  solution 
of  H2  SO4  then  add  2  or  3  c.c.  of  a 
10%  solution  of  NaOH. 

A  pink  to  red  contact  ring— Indol  re- 
action=colon. 

3rd  Metliod. 

Vide — 1,  2  and  3  of  1st  method.  Then 
apply  Riva's  1,  2  and  3  tests  to 
the  fermentation  tube  cultures 
showing  gas  formation. 

4th  Method. 

Quantitative  Examination. 

A.    1.  Plate  1  c.c,  0.5  c.c,  0.3  c.c  and  0.2 
c.c.  of  water  in  agar. 

2.  Plate  0.5  c.c.  and  0.1  c.c.  of  water 
In  agar. 

3.  Plate  a  controls  agar. 


320  BACTERIOLOGY.  1 

4.  Label  each  plate  with  the  num-1 
ber  of  the  sample,  the  quantity 
of  water  contained  and  the  date. 

5.  Incubate  at  37.5°  C. 

B.  1.  Place    9.9    c.c.     sterile  distilled 

water  in  a  sterile  capsule. 

2.  Add  0.1  c.c.  of  the  water  sample 
to  9.9  c.c.  of  water  in  the  capsule. 
This  will  give  a  dilution  of  1  in 
100. 

3.  Plate  0.5  c.c,  0.3  c.c.  and  0.2  c.c. 
of  diluted  water  in  gelatin. 

4.  Label  each  plate  with  the  quan- 
tity of  water  it  contains — that 
is,  0.005  c.c,  0.003  c.c,  and  0.002 
c.c. 

5.  Plate  0.5  c.c,  0.3  cc.  and  0.2  c.c. 
of  water  sample  in  gelatin. 

6.  Plate  a  controle. 

7.  Label  each  plate  with  the  quan- 
tity of  water  it  contains. 

8.  Incubate  at  20°  C. 

C.  1.  Plate  0.5  c.c,  0.3  c.c.  and  0.2  c.c. 

of  water  sample  in  wort  gelatin. 
2.  Label  the  plates  and  incubate  at 
20°  C. 

D.  1.  After  48  hours  incubation,  count 
^  and  record  the  number  of  colonies 

that  developed  upon  the  various 
plates. 

2.  Replace  the  gelatin  and  the  wort 
plates  in  the  incubator;  observe 
a  gain  at  3,  4  and  5  days. 

3.  Calculate  and  record  the  number 
of  organisms  present  per  cc.  of 
the  original  water  from  the  av- 
erage of  thC'  SIX  gelatine  plates 
at  the  latest  date  possible  up  to 
seven  days.  The  presence  of 
liquefying  bacteria  may  render 
the  calculation  necessary  at  an 
earlier  date,  hence  the  importance 
of  daily  observations. 

Qualitative  Examination. 

In  routine  examination  of  water,  the 
qualitative  examination  of  water  is  usu- 
ally limited  to  a  search  for  B.  Colli  and 
its  allies,  streptococci  and  some  ob- 
servers insist  on  a  search  for  the  B. 
enteritidis  sporogenes.  "The  last  organ- 
ism is  relatively  scarce  in  water,  there- 
fore, the  collection  of  a  large  quantity 
of  water  is  usually  necessary. 

During  epidemics  or  tne  examination 
of  new  and  unknown  waters,  the  coli- 
typhoid  group  are  to  be  searched  for 
and   on   occasion   the   presence   or  ab- 


BACTERIOLOGY.  321 

sence  of  vibriocholera,  B.  anthracis  or 
B.  tetani  may  need  to  be  determined. 

When  pathogenic  or  excremental  bac- 
teria are  present  in  water,  their 
number  are  few  and  it  is,  therefore,  nec- 
essary to  adopt  either  the  enrichment  or 
the  concentration  method  of  examina- 
tion. 

A.  Snrichment  Metbod.  The  harmless 
non-pathogenic  bacteria  are  de- 
stroyed or  their  growth  inhibited, 
while  the  growth  of  the  parasitic 
bacteria  are  encouraged  by  ar- 
ranging the  environment  a&  to  re- 
action of  media,  incubation  tem- 
perature and  atmosphere  so  as  to 
favor  the  growth  of  the  patho- 
genic forms  at  the  expense  of  the 
harmless  saprophytes. 
Metbod. 

1.  Number  a  set  of  Bile  salt  broth 
tubes  1-5. 

2.  Number  a  set  of  Bile  salt  broth 
tubes  la-5a. 

3.  Number  one  flask  6  and  another  7. 

4.  To  tubes  No.  1  and  la  add  0.1  c.c. 
water  sample. 

To  tubes  No.  2  and  2a  add  l.c.c. 
water  sample. 

To  tubes  No.  3  and  3a  add  2. c.c. 
water  sample. 

To  tubes  No.  4  and  4a  add  5c.c. 
water  sample. 

To  tubes  No.  5  and  5a  add  lO.c.c. 
water  sample. 

5.  Put  up  all  the  tubes  in  Buchner's 
tubes  and  incubate  aerobically 
at  42»  C. 

6.  Pipette  25  c.c.  of  double  strength 
bile  salt  broth  into  flask  6  and  50 
c.c.  double  strength  bile  salt  broth 
into  flask  7. 

7.  Pipette  25  c.c.  water  sample  into 
flask  6  and  50  c.c.  water  sample 
into  flask  7. 

8.  Incubate  the  two  flasks  aerobically 
at  42"  C. 

9.  After  the  end  of  24  hours  incuba- 
tion, note  each  culture: 

(a)  The  presence  or  absence  of  vis- 
ible growth. 

(b)  The  reaction  of  the  medium  as 
indicated  by  the  colour  change, 
if  any,  the  litmus  has  undergone. 

(c)  The  presence  or  absence  of  gas 
formation  as  indicated  by  a  froth 
on  the  surface  of  the  medium  and 


322  BACTERIOLOGY. 

the  collection  of  g-as  in  the  inner 
"gras"  tube. 

10.  Replace  those  tubes  which  show 
no  signs  of  growth  in  the  incuba- 
tor. 

Examine  after  another  period  of 
24  hours  with  reference  to  points 
indicated  above. 

11.  Remove  culture  tubes  which  show 
visible  growth  from  the  Buchner's 
tubes,  whether  acid  production  and 
gas  formation  are  present  or  not. 

12.  Examine  all  tubes  showing  growth 
by  hanging-drop  preparations. 
Note  such  as  show  the  presence 
of  chains  of  cocci. 

13.  Prepare  surface  plate  cultivations 
upon  nutrose  agar  from  each  tube 
that  shows  growth  either  macro- 
scopically  or  microscopically  and 
incubate  for  24  hours  aerobically 
at  37.5  C. 

14.  Examine  the  growth  on  the  plate 
either  with  the  naked  eye  or  with 
hand  lens.  Pick  off  for  subculti- 
vation  of  the  coll  group,  typhoid 
group,  paratyphoid  group  and  the 
streptococci.  (Practice  will  facili- 
tate the  recognition  of  the  groups). 

;15.  The  colifonn  or  tsrphiromi  colonies 
are  streak  or  smear  subcultivated 
upon  nutrient  agar  and  incubated 
aerobically  for  24  hours  at  37.5"  C. 

(a)  Examine  growth  of  each  tube 
macroscopically  and  microscopic- 
ally. 

If  growth  is  Impure,  replate  on 
nutrose  agar,  pick  olf  colonies  and 
resubcultivate  till  pure,  then  add 
5  c.c.  sterile  normal  saline  or  ster- 
ile broth  and  emulsify  the  entire 
surface  growth  with  it. 

(b)  From  the  emulsion  prepare  a 
series  of  subcultivations  by  loop 
smears  on  slanted  gelatin,  slant- 
ed agar  potato,  and  by  adding  0.1 
c.c.  of  emulsion  to  nutrient  broth, 
litmus  milk,  dextrose  peptone, 
levulose  peptone,  galactose  pep- 
tone, maltose  peptone,  saccharose 
peptone,  raffinose  peptone,  dulcite 
peptone,  marmite  peptone,  glycerin 
peptone,  inulin  peptone  and  dex- 
trin peptone. 

(c)  Differentiate  the  bacilli  by 
means  of  the  cultural  and  biologi- 
cal characters  into: 


BACTERIOLOGY.  323 

1.  SBcherlcli  gronp.  B.  Coli  commu- 
nis, B.  Coli  communior,  B.  lactis 
aerogenes  and  B.  Cloacae. 

2.  Gaertner  group:  B,  enteritidis  (of 
Gaertner),  B.  paratyphosus  A.  B. 
parathyphosus  B.  and  B.  cholerae 
suum. 

3.  Ebert  group:  B.  typhosus,  B.  dy- 
sentariae  (Shigra),  B.  dysentariae 
(Flexner),  and  B.  fercalis  alcali- 
grines. 

(d)  Confirm  results  by  specific  ag- 
glutinating- sera  obtained  from  ex- 
perimentally inoculated  animals. 
If  a  positive  result  is  obtained  by 
this  method,  it  needs  only  a  sim- 
ple calculation  to  determine  the 
smallest  quantity  of  the  sample 
that  contains  at  least  one  of  the 
organisms  of  indication,  e.g.  if 
growth  due  to  B.  Coli  in  tubes 
from  4  to  10,  it  follows  that  at 
least  one  colon  bacillus  is  pres- 
ent in  every  10  c.c.  of  the  water 
sample,  but  not  in  every  5  c.c. 


324 


BACTERIOLOGY. 


BACTERIOLOGY.  325 

16.  Pick  off  streptococcus  colonies  and 

subcultivate  upon  nutrient  agar  as 
directed  in  steps  a  and  b  of  15. 
Differentiate  the  streptococci  iso- 
lated into  members  of  the  (a) 
Saprophytic  group  —  short-chained 
cocci. 

(b)  Parasitic  (pathogenic)  group 
— long-chained  cocci  by  their  cul- 
tural characters  and  record  nu- 
merical frequency  as  indicated  af- 
ter —  of  15. 

Determine    the    pathogenicity  for 
mice  and  rabbits  of  the  strepto- 
cocci isolated. 
B.    Concentration  Metbod.  Organisms 
in  water  that  are  few  in  number 
are  best  sought  for  by  the  concen- 
tration method.    The  quantity  of 
water  required  for  this  examina- 
tion is  about  2000  c.c. 


326  BACTERIOLOGY. 


BACTERIOLOGY.  327 

Metbod. 

1.  Fit  up  filtering:  apparatus  as  in  the 
accompanying  diag-ram  (after 
Eyre). 

2.  Filter  the  entire  2000  c.c.  of  water 
through  the  filter  candle. 

3.  When  filtration  is  completed,  screw 
up  the  clamp  so  as  to  occlude  the 
two  pieces  of  pressure  tubing. 

4.  Reverse  the  position  of  the  glass 
tubes  in  Wolff's  bottle,  so  that  the 
one  nearest  the  air  pump  now  dips 
into  the  H2SO4. 

5.  Slowly  open  clamps  and  allow  air 
to  gradually  pass  through  the  acid, 
and  enter  flask,  and  so  restore 
pressure. 

6.  Unship  the  apparatus,  remove  the 
cork  from  mouth  of  candle. 

7.  Pipette  10  c.c.  of  sterile  broth  into 
the  interior  of  the  candle,  and  by 
means  of  a  sterile  test-tube  brush 
emulsify  the  slimy  residue  which 
lines  the  candle,  with  the  broth. 

Practically  all  of  the  bacteria 
contained  in  the  original  2000  c.c. 
of  water  will  now  be  contained  in 
the  10  c.c.  emulsion  of  broth,  so 
that  1  c.c.  of  emulsion  is  equiv- 
alent, so  far  as  the  contained  or- 
ganisms are  concerned,  to  200  c.c. 
of  the  original  water. 
Coll-Typlioid  Group. 

1.  Number  9  tubes  of  bile  salt  broth 
from  1-9. 

2.  To  No.  1  add  1  c.c.  of  the  original 
water  sample  before  filtration  is 
commenced. 

To  No.  2  add  2  c.c.  of  the  original 
water  sample  before  filtration  is 
commenced. 

To  No.  3  add  5  c.c.  of  the  original 
water  sample  before  filtration  is 
commenced. 

3.  To  No.  4  add  0.05  c.c.  (equivalent 
to  10  c.c.  of  original  water  sample). 
To  No.  5  add  0.125  c.c.  (equivalent 
to  25  c.c.  of  original  water  sample). 
To  No.  6  add  0.25  c.c.  (equivalent 
to  50  c.c.  of  original  water  sample). 
To  No.  7  add  0.5  c.c.  (equivalent  to 
100  c.c.  of  original  water  sample). 
To  No.  8  add  1.0  c.c.  (equivalent  to 
200  c.c.  of  original  water  sample). 
To  No.  9  add  2.5  c.c.  (equivalent  to 
500  c.c.  of  original  water  sample). 


328  BACTERIOLOGY. 

4.  Put  up  each  tube  anaerobically  in 
a  Buckner's  tube  and  incubate  at 
42"  C. 

5.  Subsequent  steps  are  same  as  those  » 
described  under  enrichment  meth- 
od. 

B.    Enteritidis  Sporogrenes. 

1.  Transfer  5  c.c.  of  emulsion  from 
the  filter  to  a  sterile  test-tube  and 
plug  carefully. 

2.  Place  test-tube  into  the  interior  of 
a  benzole  bath,  and  expose  to  a 
temperature  of  80°  C.  for  20  min- 
utes. 

3.  Number  10  tubes  of  litmus  milk 
from  1-10. 

4.  Remove  test-tube  from  benzole 
bath  and  shake  well  to  distribute 
spores  through  fluid. 

5.  Add  to  each  tube  of  litmus  milk  a 
measured  quantity  of  suspension 
vide  coli  group. 

6  Incubate  anaerobically  at  37.5**  C. 
(Put  up  in  Buchner's  tubes  or  in 
Bulloch's  apparatus,  or  pour  layer 
of  sterile  vaseline  on  surface  of 
fluid). 

7.  Examine  after  24  hours, 
(a)    Acid  reaction. 

»    (b)    Presence  of  clotting  and  sep- 
aration of  clear  whey, 
(c)    Presence  of  gas. 

8.  Replace  tubes  showing  no  signs  of 
growth  in  incubator  for  another 
24  hours  and  again  examine  vide  7. 

9.  Remove  tubes  showing  growth, 
carefully  pipette  off  whey,  and  ex- 
amine microscopically. 

10.  Inoculate  2  guinea-pigs  subcuta- 
neously  with  0.5  c.c.  of  whey  and 
observe  result. 

▼IMo  Cbolera. 

1.  Number  ten  tubes  of  peptose  water 
from  1-10. 

2.  To  each  tube  add  a  measured  quan- 
tity of  emulsion,  vide  coli  group. 

3.  Incubate  auaerobically  at  37.5"  C. 
for  24  hours  and  examine  for  deli- 
cate pellicle  formation,  which  if 
present,  examine  microscopically. 

4.  Inoculate  fresh  tubes  of  peptone 
water  from  tubes  showing  pellicle 
formation  and  incubate  for  24 
hours. 

5.  Test  peptone  water  for  nidol  and 
nitrite. 


BACTERIOLOGY.  329 

6.  Pick  off  colonies  resembling  chol- 
era colonies  and  subcultivate  on  all 
ordinary  media. 

7.  Test  vibrio  isolated  against  serum 
of  an  animal  immunized  to  cholera 
for  agglutination. 

B.  Anthrax. 

1  and  2  vide  B.  enteritidis  sporagenes. 

3.  Inoculate  a  young  white  rat  sub- 
cutaneously  with  Ic.c.  of  emulsion. 
Observe  during  life,  if  animal  dies, 
make  post  mortem  examination. 

4.  Make  nutrient  agar  plates  with 
0.2  c.c,  0.3  c.c,  and  0.5  c.c.  quan- 
tities of  suspension  and  incubate 
at  37.5''  C,  for  24  or  48  hours. 

5.  Pick  off  anthrax-like  colonies  and 
subcultivate  on  all  ordinary  media. 

6.  Inoculate  another  white  rat  as  in  3, 
using  2  loopfuls  of  agar  subculti- 
vation  emulsified  with  1  c.c.  steri^^ 
bouillon.    Observe  as  in  3. 

B.  Tetani. 

1.  Vide  1  and  2  of  B  Anthrax. 

2.  Add  1  c.c.  of  suspension  to  each 
of  3  tubes  of  glucose  formate 
broth,  incubate  anaerobically  in 
Buchner's  tubes  at  37.5**  C. 

3.  From  such  tubes  showing  visible 
growth  after  end  of  24  hours  in- 
cubation, inoculate  guinea-pigs 
subcutaneously,  using  0.1  c.c.  of 
bouillon  cultivation.  Observe  vide 
3  B  Anthrax. 

4.  From  the  same  tubes  pour  agar 
plates  and  incubate  anaerobically  in 
Bulloch's  apparatus  at  37.5"  C. 

5.  Subcultivate  suspicious  colonies  on 
various  media,  incubate  anaerobi- 
cally, making  controle  cultivation 
on  glucose  formate  agar,  stab  and 
streak,  to  incubate  anaerobically 
and  carry  out  further  inoculation 
experiments  with  resulting 
growths. 


Interpretation  of  1>acteriolosrical  water 
analysis. 

In  the  analysis  of  water,  data,  such  as 
the  kind  of  water,  the  method  of  collec- 
tion, the  sampling,  rain  fall,  transmis- 
sion, etc.,  miist  be  recorded  in  order  that 
the  results  may  be  properly  interpreted. 
Several  analyses  are  necessary  and 
should  be  made^egularly  and  systemat- 
ically. 


330  BACTERIOLOGY. 

The  number  of  micro-organisms  per- 
missible in  potable  water  depends  to  a 
g-reat  extent  upon  the  kind  of  micro- 
organisms present.  Great  numbers  of 
bacteria  indicate  a  large  amount  of  or- 
ganic matter.  The  number  of  bacteria 
in  deep  wells  and  springs  should  not  ex- 
ceed 50  per  cc.  on  gelatin  at  20°  to  22**  C. 
Organisms  in  excess  of  the  above  fig- 
ures would  indicate  pollution,  except 
after  rains  or  floods. 

The  number  of  organisms  grown  on 
agar  at  a  temperature  of  37%'*  C.  is 
probably  more  important  than  the  num- 
ber present  in  the  "gelatine  count"  in  as 
much  as  many  water  bacteria  do  not 
grow  at  this  heat,  whereas  the  sewage 
and  soil  organisms  grow  very  rapidly  at 
371/^"  C.  The  agar  count  would  there- 
fore eliminate  the  water  flora,  but 
^ould  obscure  the  sanitary  results  by 
reason  of  the  presence  of  soil  organisms. 

The  agar  count  of  deep  waters  should 
not  exceed  10  per  cc.  and  for  surface 
water  it  should  not  exceed  100  per  cc. 

Isolation  and  identification  of  specific 
disease  organisms  from  water  would 
condemn  such  a  water  as  unfit  for  use; 
bifct  by  reason  of  the  difficulty  of  such 
an  examination  their  isolation  is  not 
often  attempted. 

The  isolation  of  the  colon  bacillus 
from  water  is  easily  carried  out  and  its 
presence  is  generally  looked  upon  as 
significant  and  indicative  of  sewage  pol- 
lution. The  number  of  bacilli  coli  in  a 
certain  amount  of  water  sufficient  to 
condemn  it  varies  in  the  opinion  of  dif- 
ferent authorities.  Prescott  and  Win- 
slow  hold  that  if  present  in  1  cc.  of 
water  it  is  reasonable  proof  of  serious 
pollution.  Savage  suggests  that  the 
bacillus  coli  should  be  absent  from  10 
cc.  in  surface  waters,  such  as  rivers 
used  for  drinking  purposes,  shallow 
wells  and  upland  surface  waters. 

The  streptococcus  is  also  an  indication 
of  sewage  contamination,  and  should  be 
absent  from  the  amounts  of  water  men- 
tioned above  for  the  bacillus  coli. 

The  bacillus  enteritidis  sporogenes 
should  not  be  present  in  1,000  cc.  water 
from  deep  wells  nor  in  400  cc.  from  sur- 
face waters. 


BACTERIOLOGY.  331 

THE  BACTERIOLOGY  OF  SEWAGE. 

Sewage  is  a  menace  to  the  public 
health  because  of  the  frequent  presence 
of  pathogenic  bacteria.  It  is  made  up  of 
the  products  of  man  and  animal  waste. 
A  constant  or  characteristic  bacterial 
flora  cannot  be  established.  A  classifi- 
cation based  upon  bacterial  activity 
rather  than  upon  the  species,  the  genus, 
the  group  or  type  has  been  adopted  by 
reason  of  the  organism's  activity  in 
sewage  purification. 

Certain  exceptions  to  these  general 
principles  are  taken  in  case  of  such  or- 
ganisms as  the  bacillus  coli,  the  sewage 
streptococcus  and  the  bacillus  enteri- 
ditis.  These  are  to  a  certain  extent 
characteristic  sewage  bacteria  and  their 
interest  in  them  as  individuals  has  to  do 
with  water  bacteriology. 

According  to  the  general  character  of 
changes  which  the  sewage  organisms 
bring  about  they  are  divided  into  two 
large  groups  as  follows: 

(1)  The  anserobic  or  putrefactive 
bacteria  which  bring  about  the 
withdrawal  of  oxygen  from  one 
molecule  or  part  of  a  molecule 
and  the  subsequent  oxidation  of 
another  molecule  or  part  of  the 
same  molecule.  The  energy  re- 
leased in  this  process  is  utilized 
in  the  vital  functions  of  the  or- 
ganisms. It  involves  the  reduc- 
tion of  urea,  the  hydrllis  of  pro- 
tein, and  of  cellulose,  the  emul- 
slfication  of  fats,  the  reduction  of 
nitrates  and  sulphates  and  pos- 
sibly phosphates. 

(2)  The  Ozidizingr  Bacteria.  They 
are  distinguished  by  the  fact 
that  oxygen  is  added  to  the 
molecule,  the  product  containing 
more  oxygen  than  the  initial 
substance.  Carbon  dioxide,  water 
and  nitrates  are  produced  in 
distinction  from  methane  hy- 
drogen and  ammonia  which 
characterizes  the  anserobic  re- 
actions. 

Fathogfenic  Bacteria.  It  is  assumed 
that  they  are  always  present  in  sewage. 
Sewage  can  be  made  harmless  by  being 
sterilized  but  can  be  freed  from  offense 
only  by  the  destruction  of  organic  mat- 
ter, and  this  is  obtained  almost  wholly 


332  BACTERIOLOGY. 

through  bacterial  processes,  except  when 
chemical  precipitants  are  used. 

There  are  two  general  methods  em- 
ployed for  the  cultivation  of  those  bac- 
teria which  are  of  assistance  in  sewage 
purification.  They  may  be  cultivated  in 
niters  of  sand  or  coarser  material,  or  in 
specially  constructed  tanks  called  "sep- 
tic tanks." 

Septic  Tank  Furification. 

Cameron,  1895,  introduced  the  "septic 
tank."  In  this  tank,  the  sewage  was  ad- 
mitted at  the  bottom  and  flowed  out  at 
the  top  after  about  24  hours'  subjection 
to  anaerobic  conditions  acting  upon  the 
organic  matter  as  indicated  above.  Soil 
and  sand  filters  act  not  only  mechan- 
ically, but  also  bacteriologically  and 
ofCer  one  of  the  best  means  of  purifying 
sewage  bacteriologically.  Sewage  is 
conducted  to  beds,  allowed  to  pass 
through  and  then  after  a  few  hours 
again  poured  on.  This  purification  is 
produced  by  the  action  of  aerobic  bac- 
teria. The  best  results  are  obtained  by 
combining  the  two  processes;  first,  the 
anaerobic  treatment  to  break  down  the 
solid  material,  then  the  sand  filtration  to 
oxidize  the  compounds  and  render  these 
products  harmless. 

The  biological  processes  remove  bac- 
teria not  by  any  specific  antagonistic 
action  but  by  delaying  their  passage  and 
permitting  the  natural  decrease  that  oc- 
curs when  multiplication  is  prevented. 

Sewagre  Analysis. 

On  account  of  the  great  numbers  of 
bacteria  in  sewage  it  becomes  neces- 
sary to  make  dilutions  ranging  from 
1-1000  to  1-10000  and  then  plating, 
etc.,  as  in  water  analyses. 
In  sewage  chemistry,  putrefaction  Is 
that  change  which  takes  place  naturally 
in    sewage    after    anaerobic  conditions 
have   become   established.    It  involves 
the  reduction  of  urea,  the  hydrolysis  of 
protein  and  of  cellulose,  the  emulsifica- 
tion  of  fats,  the  reduction  of  nitrates 
and   sulphates   and   possibly   of  phos- 
phates and  those  other  changes  which 
are  characterized  by  the  withdrawal  of 
O  and  the  hydrolysis  of  complex  mole- 
cules.   These  changes  are  always  noted 
in   sewage   under   anaerobic  conditions, 
and  the  terms  putrefactive  and  anaerobic 
change  are   for   the  present  purposes 
practically  synonymous. 


BACTERIOLOGY.  383  ^ 

BACTERIOLOGY  OF  SOIL 

Many  varieties  of  bacteria  are  pres- 
ent in  the  soil.  Some  by  reason  of  con- 
tamination through  animal  feces  and 
other  waste  products,  but  the  majority 
are  real  soil  bacteria  in  that  they  live 
and  multiply  chiefly  in  the  soil.  They 
have  important  functions*  to  preform  in 
continuing-  the  earth's  supply.  Some  of 
the  bacteria  make  carbon,  nitrogen,  hy- 
drogen and  other  compounds  locked  up 
in  the  dead  bodies  of  animals  and 
plants  available  for  plants.  Other  bac- 
teria manufacture  food  for  plants  from 
the  gases  of  the  air  and  the  inorganic 
elements  of  the  earth,  which  in  their 
simple  forms  were  not  available.  They, 
therefore,  form  an  important  link  in  the 
earth's  life  cycle.  Food,  moisture  and 
proper  temperature  are  necessary  for 
their  activities. 

In  a  grain  of  rich  loam  there  may 
be  many  millions,  while  an  equal  quan- 
tity of  sand  may  be  almost  free  from 
bacteria. 

Various  species  of  soil  bacteria  have 
an  influence  upon  each  other.  The  an- 
aerobic bacteria  are  enabled  to  develop 
by  the  aerobic  species  utilization  of  the 
free  oxygen,  while  still  other  species 
make  assimilable  substances  which  can- 
not be  used  by  others. 

Carbon  compounds  are  broken  up  by 
the  soil  bacteria.  Starch  is  manufac- 
tured by  plants  then  converted  into  cel- 
lulose, wood  fats  and  sugar,  which  when 
formed,  cannot  be  utilized  by  other 
plants. 

The  largest  part  of  these  substances 
are  broken  up  by  micro-organisms;  a 
smaller  portion  are  transformed  within 
the  animal  body.  Alcohol  is  fermented 
from  sugars  and  starch  by  the  yeasts 
and  molds  with  the  production  of  carbon 
dioxide,  or  acid  fermentation  by  the 
action  of  bacteria  takes  place,  with  the 
production  of  acids  and  often  of  carbon 
dioxide. 

Cellulose  is  attacked  by  certain  vari- 
eties of  bacteria,  acting  both  In  the 
presence  and  absence  of  free  oxygen. 
Moulds  also  act  on  cellulose  with  pro- 
duction of  carbon  dioxide,  gas  and  other 
products.  Wood  is  first  attacked  by 
fungi,  then  by  bacteria. 

Animals  utilize  plant  proteids  and  re- 
duce  them   to   simpler   compounds  as 


384  BACTERIOLOGY. 

urea,  etc.,  but  these  compounds  are  not 
suitable  for  plant  use,  so  that  micro- 
organisms must  break  tnese  compounds 
into  more  simple  form.  Yeasts,  molds 
and  fungi  decompose  these  substances 
(plant  proteids)  to  a  certain  extent,  but 
the  chief  decomposition  is  carried  out  by- 
bacteria. 

In  the  absence  of  oxygen,  the  process 
is  incomplete  with  the  production  of 
H2S,  NH3  and  CH4,  and  is  termed  putre- 
faction. The  presence  of  oxygen  gives 
rise  to  more  complete  decomposition 
with  the  production  of  CO2,  N  and  H2O. 

The  variety  of  organisms  producing 
these  changes  are  many.  Some  are 
found  in  decaying  vegetable  matter, 
others  in  animal  tissue. 

There  Is  a  process  of  oxidation  pro- 
duced by  bacteria,  in  which  ammonia 
compounds  are  changed  to  nitrates  and 
thus  utilized  by  plants  (nitrification). 
The  ammonia  is  first  oxidized  to  nitrite, 
then  into  a  nitrate,  which  is  taken  up 
by  the  plant  roots  from  the  soil.  The 
two  organisms  isolated  causing  a  change 
of  ammonia  to  nitrites,  are  the  nitroso- 
menas  and  the  nitrosococcus.  One  vari- 
ety of  organism  changing  nitrites  to 
nitrates  is  called  nitrobacter.  These 
organisms  appear  to  depend  upon 
mineral  substances  for  their  food. 
A  small  amount  of  organic  matter  in 
the  media  acts  as  antiseptics.  Plants 
take  up  most  of  their  nitrogen  in  the 
form  of  nitrates;  hence  these  bacteria 
are  important.  When  the  soil  becomes 
acid,  the  growth  ceases.  Air  is  neces- 
sary for  their  action  as  the  process  is 
one  of  oxidation. 

There  is  also  a  reduction  process 
called  dentrification.  Nitrates  yield  a 
part  or  all  of  its  oxygen  and  becomes 
changed  to  nitrites,  ammonia,  and  free 
nitrogen. 

In  the  partial  change,  the  soil  does 
not  lose  its  available  nitrogen  which 
takes  place  in  the  total  change,  by 
changing  nitrites  and  ammonia  by  the 
nitrifying  bacteria  to  nitrates. 

The  types  of  nitrogen  reduction  are: 

1.  The  reduction  of  nitrates  to  nitrites 
and  ammonia. 

2.  The  reduction  of  nitrates  and  ni- 
trites to  gaseous  oxides  of  nitrogen. 

3.  The  reduction  of  nitrites  with  the 
development  of  free  nitrogen. 


BACTERIOLOGY.  386 

Certain  plants  are  able  to  use  the  ni- 
trogen of  the  air  through  the  aid  of 
bacteria  growing  in  and  producing  en- 
largments  (tubercles),  on  the  roots.  The 
root  bacteria  are  called  B.  radicicola  and 
may  remain  active  in  the  soil  for  long 
periods  even  though  there  is  no  legu- 
minous vegetation. 

The  organism  diffuses  rapidly  in  soils 
that  are  in  proper  conaition,  so  that  if 
a  soil  lacks  the  organism,  it  cannot  be 
introduced  to  it  until  the  soil  has  been 
made  fit  for  the  organisms  development. 
Buchanan  concludes  that: 

1.  The  B.  radicicola  varies  considera- 
bly in  its  morphology  when  appro- 
nutrients,  as  the  salts  of  organic 
acids,  are  induced  into  the  artifi- 
cial media.  Sodium  succinate  pro- 
duces a  most  luxuriant  growth  to- 
gether with  the  greatest  variety  of 
bacteroids. 

2.  The  B.  radicicola,  in  the  roots  of 
legumens,  inay  show  the  same  type 
of  bacteroids  as  seen  in  suitable 
artificial  media,  and  again  the  same 
type  may  not  be  the  same  as  pro- 
duced in  culture-media  and  that 
produced  in  the  nodule  by  the  same 
form. 

3.  The  B.  radicicola  probably  includes 
a  group  of  closely  related  varie- 
ties or  species  which  differ  from 
each  other  and  morphological  char- 
acters. 

4.  The  organism  of  the  nodule  re- 
sembles morphologically  both  the 
yeasts  and  the  bacteria.  The  dif- 
ference between  this  form  and  the 
forms  included  ander  bacillus  and 
pseudomonas  justify  the  generic 
use  of  a  separate  generic  name  of 
Rhizobium. 

Winogradsky  states  that  certain  an- 
aerobic, spore-bearing,  bacilli  (Clostri- 
dium Pasteurianum)  outside  of  the  roots 
perform  the  same  function  as  those 
within  the  roots.  Their  power  of  nitro- 
gen fixation  is  increased  in  the  presence 
of  sugar  and  decreased  in  the  presence 
of  nitrogenous  substances. 

Beyerinck  and  Bailey  have  described 
aerobic  species  of  nitrogen  fixing  bac- 
teria, to  which  the  name  of  Azotobacter 
has  been  given. 

The  inoculation  of  soils  and  an  in- 
vestigation of  soils  and  crops  best  fitted 


38«  BACTERIOLOGY. 

for  the  growth  of  these  bacteria  has 
been  carried  out  with  the  result  of 
greatly  Improving  inpoverished  soils. 

The  use  of  seeds  inoculated  with  a 
special  variety  of  bacteria  suitable  for 
the  plant  and  soil  has  been  largely 
practiced  with  marked  results.  Exces- 
sive bacterial  development  may  at  times 
be  harmful  to  the  soil. 

The  exhaustion  of  the  soil  following 
the  constant  raising  of  the  same  crop  is 
now  thought  to  be  due  partly  to  the 
inability  of  a  few  restricted  species  of 
bacteria,  continued  in  the  soil,  to  pro- 
duce the  substances  necessary  for  the 
nutrition  of  the  special  crop,  or  that  the 
bacteria  use  up  the  substances  in  the 
soil  necessary  to  crop  growth.  j 

The  greatest  number  of  bacteria  are 
found  a  little  below  the  surface  of  the 
soil. 

Some  of  the  bacterial  products  act 
upon  the  inorganic  constituents  of  the 
soil.  The  CO2  and  organic  acids  act  up- 
on the  compounds  of  lime  and  magnesia, 
and  convert  them  into  more  soluble  sub- 
stances. The  same  is  true  of  the  rock 
phosphates,  the  silicate  of  potassium, 
sulphates,  etc. 

Quantitative  Analysis  of  soil  for  bacteria 

Include  4  distinct  investigations: 

1.  Enumeration  of  Aerobic  organism. 

2.  Enumeration  of  Spores  of  Aerobes. 

3.  Enumeration  of  Anaerobic  organ- 
isms (also  facul.  Anaerobes). 

4.  Enumeration  of  Spores  of  organ. 
Further  by  a  combination  of  results  , 

of  1-2  and  3-4,  the  ratio  of  spores 
to  vegetative  forms  is  obtained. 

1.  Obtain  soil  under  sterile  conditions. 

2.  Weigh  and  make  proper  dilutions 
for  counting. 

3.  (A)  Aerobe— 

Pour  set  of  gelatin  plates. 
Incubate  at  20°  C. 
Pour  set  of  agar  plates. 
Incubate  at  371/2"  C. 

4.  Count  plates  for  3,  4  or  5  days. 

(a)  The  number  of  aerobic  micro 
organisms  per  1  gm.  soil. 

(b)  The    number    of    yeast  and 
moulds  per  1  gm.  soil. 

(c)  The  number  of  aerobe  growing 
at  37"  C,  per  1  gm.  soil. 


BACTERIOLOGY.  837 

(B)  Anaerobes  spores  and  Vegr. 
Pour  set  of  plates  in  glucose. 
Formate  gelatin  and  agar. 
Incubate  in  Bulloch's  apparatus. 

(C)  Aerohes  and  Anaerobes  (spores 
only). 

(1)  5  c.c.  of  soil  dilution  in  sterile 
tube. 

(2)  Differential  sterilize  at  80  for  10 
minutes. 

( 3  )  Pour  plates  and  incubate  anaerobi- 
cally. 

(4)  After  long  incubate,  count. 
Qualitative — f  or 

Coli  group — Typhoid  group. 

B.  Anthrax,   B.   Tetanus,   B.  Malig 

Oedema. 

Nitrous  Organism,  Nitric  Organisms. 
Nitrous  Orgran. 

10  tubes  of  Winogradsky's  Sol.  No.  1. 

Label  from  1-10. 

Inoculate  each  tube  with  varying 
dilutions  and  incubate  at  30**  C. 
Nitric  Organisms. 

10  tubes  of  Winogradsky's  Sol.  No. 
11,  and  incubate  as  in  1. 
Incubate  at  30"  C. 
Examine  after  24-48  and  from  those 
tubes  that  show  signs  of  growth  make 
subcultivations  in  fresh  tubes  of  same 
media   and   incubate  at   30"   C.  Make 
further  subcultivation  from  these  and 
again  incubate. 

If  growth  occurs  in  these  sub-cul- 
tures, make  surface  smears  on  plates  of 
Winogradsky's  silicate  Jelly.  Pick  off 
colonies  as  make  appearance  and  sub- 
cultivate  in  each  of  these  two  media. 
W.  Sol.  for  Nitric. 

1.  K.  Phosphate,  1  gm. 
Mg.  Sulph.,  .5  gm. 

Ca.  Chloride,  .01  gm. 
Na  Chlor,  2  gm. 
Dissolve  in  A.D.,  1000  c.c. 

2.  Fill  into  flasks  in  quantities  of  20 
c.c,  and  add  to  each  a  small  quan- 
tity of  freshly  washed  mg.  Carb. 

3.  Sterilize  in  steamer  at  100  for  3 
days. 

4.  Add  to  each  flask,  2  c.c.  of  sterile 
2%  sol  Ammonia  Sulph. 

5.  Incubate  at  37°  for  48  hours  and 
eliminate  any  contaming  a  growth. 

Tlie  Media  for  the  growth  of  Nitros 

Organisms. 
1.  Ammon.  Sulph.,  1  gm. 

K.  Sulph.,  1  gm. 

A.D.,  1000  c.c. 


838  BACTERIOLOGY. 

2.  Add  5-10  gm.  basic  mg".  Garb  (ster- 
ilize by  boiling:). 

3.  Pill  flasks  and  sterilize  as  in  No.  1. 
W.  SUicate  JeUy. 

Sol.  A. 

Ammon   Sulph.,   40   gm.  Mgr. 

Sulph.,  0.05  gm. 
Calcin  Chloride,  .01  gm.,  A.D. 

50  c.c. 

Sol.  B.    K.  Phosphate  .10  gm.  Na  Carb 
.60  gm.  A.D.  50  c.c. 
Silica  acid   3.4   gm.,  A.D.   100  c.c. 
Pour  them  into  a  large  dish,  (Por- 
celain). 

5.  Mix  of  Sol.  A  &  B  then  add  suc- 

cessive small  quantities  of  mixed 
salts  to  silicic  acid  sol.  (stir  const.) 
.  with  glass  rod  till  a  Jelly  of  right 
consistency  is  found. 

6.  Spread  layer  of  Jelly  over  several 
Petri  dishes. 

Sterilize  for  30  minutes  on  3  days. 

THE  BACTERIOLOGY  OF  AIR. 

The  atmosphere  is  not  the  normal 
habitat  of  bacteria.  Their  growth  and 
multiplication  can  not  take  place  in  it 
under  ordinary  conditions.  The  air  is 
kept  in  motion  by  the  wind,  so  that  fine 
pa»rticles  are  constantly  being  carried 
into  it  from  the  ground,  especially  so  in 
inhabited  areas.  The  bacteria  in  the 
dust  of  the  field  and  street  are  carried 
along  with  the  dust  particles  of  the  air 
and  are  usually  of  the  harmless  soil 
variety  ©r  the  almost  harmless  intes- 
tinal bacteria  of  animals.  Pathogenic 
human  bacteria  are  rarely  carried  in 
harmful  numbers  except  under  excep- 
tional circumstances,  and  are  usually  in 
form  of  spores,  e.  g.,  anthrax  bacillus, 
tetanus  bacillus.  On  a  dry,  windy  day 
the  air  contains  many  thousands  of  bac- 
teria per  cubic  meter.  In  warm  weather 
the  rain  carries  down  the  bacteria  of  the 
air.  After  a  storm  there  are  very  few 
bacteria  present  in  the  air.  The  bac- 
teria in  the  air  of  the  country  are  much 
less  than  the  bacteria  in  the  air  of  the 
cities.  Forests  decrease  the  number  of 
bacteria.  Bacteria  are  very  few  on  high 
mountains;  also  on  the  high  seas.  The 
bacteria  that  multiply  in  the  streets  and 
in  the  soil  are  almost  always  sapro- 
phytic. The  bacteria  present  in  the  air 
of  dwellings  depend  upon  factors  such 


BACTERIOLOGY.  339 

as  the  opening  of  windows  to  the  out- 
side, the  cleanliness  of  the  dwellingr  and 
the  stirring  up  of  dust  t)y  sweeping.  It 
is  nearly  impossible  to  separate  the  ef- 
fects of  the  bacteria  which  we  inhale 
from  that  of  the  dust  particles  which 
they  accompany.  Both  probably  act  as 
slight  irritants  and  so  predispose  to 
definite  infections. 

It  is  problematical  as  to  the  impor- 
tance of  air  as  a  means  of  conveying  dis- 
ease, though  there  can  be  no  doubt  that 
smallpox,  measles,  scarlet  fever,  etc., 
are  transmitted  readily,  and  pulmonary 
anthrax  and  tuberculosis,  pneumonia, 
influenza,  diphtheria  and  meningitis 
may  result  from  inhalation  of  the  or- 
ganisms. 

The  distance  through  which  the  air 
may  carry  the  causative  agents  of  dis- 
ease requries  further  study  and  must 
necessarily  depend  upon  a  variety  of 
conditions,  as  time,  degree  of  moisture, 
air  currents,  factors  producing  desicca- 
tion, effects  of  sunlight,  etc. 
Bacterial  Air  Examination. 

Gelatin  or  agar  plates  may  be  exposed 
to  the  air  for  a  definite  length  of 
time  and  then  incubated  at  both 
25°  and  37%°  C.  temperatures. 

The  number  of  colonies  appearing  on 
the  plates  will  indicate  in  a  general  way 
the  bacterial  content  of  the  air.  The 
results  must  necessarily  vary  according 
to  the  degree  of  moisture  in  the  atmos- 
phere, air  currents,  etc.,  and  therefore 
furnishes  no  standard  for  comparative 
results. 

A  method  in  use  at  the  present  time, 
from  which  more  accurate  results  may 
be  obtained,  follows,  vide: 

1.  Pill  10  litres  of  water  into  an  aspi- 
rating bottle. 

2.  Construct  a  sand  filter  from  a 
small  glass  tube  qontaining  about  4 
cm.  depth  of  quartz  sand  held  in 
place  by  means  of  a  wire  screen. 
Sterilize  in  hot  air  oven. 

3.  Insert  sand  filter  in  a  perforated 
rubber  stopper  which  fits  mouth  of 
aspirating  bottle. 

4.  By  allowing  water  to  flow  from 
lower  opening  of  aspirating  bottle, 


340  BACTERIOLOGY. 

10  litres  of  air  is  now  drawn 
through  the  sand  niter.  (Refill  as- 
pirating: bottle  and  aspirate  as 
many  times  as  necessary  to  give  the 
quantity  of  air  required  for  the 
test.) 

5.  Remove  the  sand  filter  and  care- 
fully pour  the  sand  into  a  known 
quantity  of  sterile  bouillon  or  water. 

6.  From  the  bouillon-sand  mixture 
(which  contains  in  suspension  the 
total  number  of  bacteria  contained 
in  the  quantity  of  air  aspirated 
through  the  sand)  plate  1  c.c.  in  gel- 
atin or  agar  and  from  the  colonies 
appearing  thereon  estimate  the 
number  of  bacteria  contained  in  a 
given  quantity  of  air. 

By  the  use  of  the  aspirating  bottle  the 
air  may  be  drawn  directly  through  a 
measured  quantity  of  sterile  bouillon  or 
water  in  an  Erlenmeyer  flask  as  follows: 

(1)  Erlenmeyer  flask  of  250  c.c.  ca- 
pacity containing  50  c.c.  Bouillon 
or  water. 

(2)  Rubber  stopper  to  fit  mouth  of 
flask  perforated  with  2  holes  and 
fitted  as  follows:  Take  a  9  cm. 
length  of  glass  tubing  and  bend  up 
3  cm.  at  one  end  at  right  angles 
to  main  length.  Pass  long  arm  of 
the  angle  through  one  of  the  per- 
forations in  the  stopper.  It  must  not 
come  in  contact  with  the  bouillon. 

Take  a  glass  funnel  5  or  6  cm.  in 
diameter,  with  a  stem  12  cm.  in  length, 
and  bend  stem  close  up  to  the  apex  of 
the  funnel,  in  a  gentle  curve  through  a 
quarter  of  a  circle;  pass  the  long  stem 
through  the  other  perforation  in  the 
rubber  stopper.  Make  sure  the  end  of 
the  stem  of  the  funnel  is  immersed  in 
the  bouillon. 

(3)  Sterilize  flask,  contents  and  fit- 
tings. 

(4)  Attach  aspirating  bottle  to  small 
glass  tube  and  operate  as  in  "4"  of 
sand  filter. 

(5)  Maae  plates  from  bouillon  as  in 
"6"  of  sand  filter. 


INDEX 


Adiorion  Schoen- 

leinii,  279. 
Acid  fast  bacteria, 
86. 

group  of  organ- 
isms, 229. 
production  test 
for,  89,  90. 

Actinobacillosis, 
277. 

Actinomyces, 
86,  275. 

Active  stage  of 
bacteria,  28. 

Aerobes,  16. 

Aerogenio  bacteria 
34. 

Agglutinin, 

124,  154,  155. 

Agressin,  178. 

Agar  agar,  41. 
sulphindigo- 
tate,  45. 

Alcoholic  produc- 
tion, test  for,  92. 

Alexin,  123,  129. 

Amboceptor, 
129,  143. 
test  for,  136. 

Ammonia  produc- 
tion, test  fon# 
91.  ^ 

Ammonification, 
18. 

Amylases,  12. 

Amylolytic  fer- 
ments, 12. 

Anaphylaxis,  183. 

Anabolic 

activities,  17. 

Anaerobes,  16. 

Anaerobic  cultiva- 
tion, 63. 
cultivation 
methods,  63. 

Analyses  of  soil, 
336. 

Anthrax,  209. 
vaccine,  166. 


Anthrax-like 
bacilli,  213. 

Animal  inocula- 
tion, 110. 

Anterior  poliomy- 
litis,  290. 

Antiamboceptor, 
135. 

Antibacterial 
serum,  34. 

Antibodies,  12,  123. 
determination 
of,  138. 

Anticomplements, 
136. 

Antidysenteric 

serum,  165. 
Antiferments,  12. 
Antigens,  124,  141. 
Antigonococci 

serum,  164. 
Antiseptics,  test 

for,  103. 
Antistreptoccic 

serum,  164. 
Antitoxins,  12,  123, 

159. 

Antitoxic  serum, 
34. 

Articular  rheuma- 
tism, 289. 

Aromatic  producits, 
14. 

Anthrogenous 

spore  forma- 
tion, 30. 

Asparagin  Uschin- 
sky's,  45. 

Asporogenousi  bac- 
teria, 30. 

Asiatic  cholera, 
225. 

cholera  vaccine, 
173. 

Atmospheric  opti- 
mum, test  for, 
96. 

Autolysin,  133. 
Azotobacter,  21. 


BaoUluB  aerosrenes, 

260. 

aerogenes  capsu- 
latus,  246. 

alkaligenes, 
263,  266. 

amethystinus, 
310. 

auranticus,  310. 
anthracis,  209. 
anthracoides, 

214. 
blue  pus,  203. 
Bordet-&eng^on, 

222. 

Botulinus,  248. 
Bovis  morbifi- 

cans,  265. 
bubonic  plague, 

250. 

butyricus,  237. 
chicken  cholera, 
253. 

cholera  sins,  266. 
circulans,  310. 
cloacae,  311. 
coerulens,  310. 
coli,  256. 
coli  communior, 
'  259. 

©oli  communis, 
256. 

coli  groi^p  of  or- 
ganisms, 256. 

diphtheria,  214. 

Ducrey,  224. 

dysenteria 
Shiga,  267. 

group  of  organ-^ 
isms,  267. 

enteritidis,  263,. 
264. 

enteritidis 
sporagenes, 
248,  328. 

fluorescence 
aurens,  310. 

fluorescence 
crassus,  310, 

fluorescence 
longus,  310. 

fluorescence 
liquefacieus 
group    of  or- 
ganisms, 310. 


fluorescence  non 
liquefacieus 
group   of  or- 
ganisms, 310. 

fluorescense  ten- 
nis, 310. 

fulous,  310. 

green  pus,  203. 

glanders,  206. 

hog  cholera,  266. 

Hoffmanni,  218. 

mdicus,  310. 

influenza,  219. 

ioteroides,  266. 

janthinus,  310u 

leprosy,  237. 

leprosy,  rat,  237. 

liquefacieus,  310. 

lividus,  310. 

Lustgarten,  237. 

malignant  ode- 
ma,  245. 

mallei,  206. 

megathesium, 
310. 

mesentericus, 
310. 

mirabilis,  310. 

Morax-Axenfeld, 
223. 

mucosus  capsu- 

latus,  261. 
murisepticus, 
222. 

mycoides,  310. 

ochracens,  310. 

ozena,  261,  263. 
jparadysenteria 
^.  "A"  Parke, 
269. 

paradysenteria, 
**B"  Flexner, 
269. 

paratyphoid,  266. 

pleuro-pneu- 
monia  of  rab- 
bits, 222. 

prodigiosus,  310. 

proteus  vulgaris, 
205. 

psittacosis, 
264. 

radicicolus,  20. 
radicosus,  214., 
rat  leprosy,  240. 


rhinoscleroma, 

261,  263. 
rhusiopathlar, 

222. 

rubefacens,  310. 

ruber,  310. 

rubescens,  310. 

smegmatio,  237. 

soft  chancre,  224. 

subtilis,  310,  314. 

swine  plague, 
254,  267. 

symptomatic  an- 
thrax, 242. 

tetani,  240. 

timothy,  236. 

tuberculosis,  229. 

tuberculosisv  av- 
ian, 235. 

tuberculosis,  bo- 
vine, 235. 

typhimurium, 
265. 

typhosus,  270. 
typhosus  g-roup 

of  organisms, 

270. 

violaceus,  310.  . 

vulgaris,  311. 

whooping  cough, 
222. 

xeroses,  219. 

Zenkeri,  311. 

Zopfii,  311. 

Zurneeden,  224. 
Bacilli  resembling 

bacillus  of  tub- 
erculosis, 236. 
Bacteria  in  air,  5. 

in  body,  6. 

in  foods,  5. 

in  soil,  5. 

in  water,  5. 

normal  to  human 

body,  6. 
Bacteria  reaction 

to  stains,  72. 
Bacteriaceae,  23. 
Bacterial  cultiva- 
tion, 61. 

enzymes,  11. 

ferments,  11. 

growth  charac- 
teristics, 67. 

identification,  65. 

mobility,  27. 

nutritian,  15. 


proteins,  11. 

sheath,  27. 
Bacteraemia,  118. 
Bacterins,  174, 
Bacteriology  of  air 
338. 

of  milk,  304. 

of  sewage,  331. 

of  soil,  333. 

of  water,  309. 
Bacteriolysins, 
124. 

Bacterium  avisep- 
ticus,  253. 
boviseptium, 
255. 

diphtheria,  214. 
Bacterium  lactus 
aerogenes,  260. 
pneumonia,  261. 
suisepticus,  254. 
tularense,  253. 
Beri-beri,  289. 
Beyrinck's  media, 

49,  50. 
Bile  salt  broth,  45. 
Biochemical  mejth- 

ods,  14. 
Biology     of  bac- 
teria, 5. 
Biologic  activitiea) 
of   bacteria,  17. 
classification  of 
bacteria,  32. 
Black  death,  250. 
Black  leg,  242. 
Blastomycetes, 
281. 

Blood  agar,  43. 
Blood  serum,  42. 
Bouillon,  41. 
Branched  baciteria, 
25. 

Bubonic  plague, 
250. 

Bubonic  plagn© 

vaccine,  173. 
Buffon,  3. 

Calcium  cycle,  22. 
Capsule    of  bac- 
teria, 26. 
Carbon  cycle,  21. 
Casease,  13. 
Cell  membrane,  26. 

wall,  26. 

content,  27. 


Chalamydobax;- 
teriaceae,  28. 

Chalamydozoa, 
294. 

Chemical  constitu- 
ents of  bacterial 

cell,  15. 
Chicken  cholera, 

253. 

Chromogenic  bac- 
teria, 33. 

Chromparous  bac- 
teria, 33. 
gram  negative 
cocci,  201. 

Cladothrix,  274. 

Classification  of 
bacteria,  22. 

Clostridium,  21. 

Coagulating  fer- 
ments, 13,  32. 

Coccaceae,  28. 

Cohn,  4. 

Colon-typhoid  dys- 
entery, group 
of  bacilli,  256, 

Coma  bacillus  of 
Koch,  225. 

Complement,  129, 
144,  150. 
fleviation,  136. 
fixation  of,  136, 

139,  140. 
method  of  fixa^ 
tion,  140. 

Contagious 

pleuro-pneu- 
monia  of  cat- 
tle, 294. 

Cornstalk  disease, 
255. 

Copula,  129. 

Cultivation  of  bac- 
teria, 36,  61. 

Culture  media,  36, 
37. 

Davaine,  5. 

Degeneration 

forms  of  bac- 
teria, 26. 

Denitrification,  20. 

Dextrose  bouillon, 
44. 

Diastases,  12,  32. 
differential 
staining,  78. 


Dilutions  in  culti- 
vation, 62. 

Diplococcus  gonor- 
rhoea, 197. 
lanceolatus,  192. 
mucosus,  201. 
pneumonia,  192. 

Diphtheria,  214. 

Diptheretic  anti- 
toxins, 161. 

Diseases  produced 
by  higher  bac- 
teria, 274. 
of  unknown  eti- 
ology, 283. 

Disinfectants,  test 
of,  103. 

Dissolving  fer- 
ments, 13. 

Distribution  of 
bacteria,  5. 

Dorlset's  egg  me- 
dia, 43. 

Dunham's  inosite- 
free  bouillon, 
46. 

Dysentery,  267. 


Ebert     group  of 
organisms,  ' 
323. 

Ectoplast,  27. 

Egg  media,  44. 

Ehrlich,  5. 

Eisenberg's  rice- 
milk  media,  48. 

Endogenous  spore 
formation,  29. 

Endotoxins,  119. 

Enzymes,  11. 

Enzyme  produc- 
tion, 87,  88. 

Erhenberg,  3. 

Eschrich  group  of 
organisms,  323. 

Elubacteria,  23. 

Eumycetes,  278. 

Extracellular  tox- 
ins, 34,  109. 


Facnltative  anaer- 
obes, 16.  35. 

Pat  splitting  fer- 
ments, 13. 

Pavus,  279. 


Ferment  at  ion,  12, 
32. 

Ferments,  12. 

amylolytic,  12. 

coagulating",  13. 

effects  of,  14. 

emulsifying,  13. 

inverting,  13. 

lypolytic,  13. 

oxidases,  13. 

proteolytic,  12. 

reducing,  14. 

ureases,  13. 
Filtering  media, 

40,  60. 
Flagellar  motility, 
27. 

Flexner's  bacillus, 
269. 

Foot  and  mouthi 
disease,  291. 

Formation  of  gon- 
idia,  31. 

Fracas  t  or,  3. 

Friedlander's  bac- 
illus, 261. 
group  of  organ- 
isms, 261. 

Frombresia  trop- 
ica, 304. 

Fusiform  bacillus, 
297. 

G-artner's  bacillus, 
264. 

group  of  organ- 
isms, 323. 
Gas  production, 

test  for,  95. 
Gelatin,  41. 

stab  culures,  68. 
Genito-urinary 

tract  flora,  10. 
Glanders,  206. 
Gonidia,  31. 
Gonococcus,  197. 
Growth  conditions 
for  bacteria, 
35. 

Haemorliaglc  sep- 
ticemia group 
of  organisms, 
250. 

Haemolytic  serum, 
130,  143. 
unit,  143. 


Haemolysin,  129, 
132. 

Hanging  drop,  71. 

Haptins,  124. 

Hay  bacillus,  214. 

Heller's  urine 
agar,  48. 

Hesse's  method  of 
anaerobic  cul- 
tivation, 63. 

Heterolysis,  132. 

Higher  bacteria, 
25. 

History  of  bacter- 
iology, 3. 

Hog  cholera,  266. 
vaccine,  169. 

Hydrophobia,  286. 

Hydrolytic  fer- 
ments, 13. 

Hypersusceptibil- 
ity,  183. 

Hyphomycetes, 
278. 

Immtinity,  study 

of,  108,  120. 
Immune  body,  120. 

test  for,  136. 
Immunization, 

study  of,  126, 

127,  128. 
Indol  production, 

test,  93. 
Infection,  114,  116, 

118. 

blood  examina- 
tion methods 
in,  115. 
conditions  ne- 
cessary 'to, 
116,  117. 
general  observa- 
tion of,  115. 
special  observa- 
tion of,  115. 
Infectious  dis- 
eases, 33. 
Infection  trans- 
mitted by  milk, 
307. 
Influenza,  219. 
Influenza-like  bAr 

cilli,  221. 
Inoculation,  llOu 
materials  used, 
111. 


methods  of,  111, 
112,  113. 
Inseperate  toxins, 
34. 

Intestinal  flora,  8. 

Intracellular  tox- 
ins, 34,  109. 

Invertases,  13,  32. 

Inverting"  fer- 
ments, 13. 

Involution  forms 
of  bacteria,  26. 

Isolation  of  bac- 
teria, 107. 

Isolysins,  133. 

Kata.l>olic  activi- 
ties of  bac- 
teria,   10,  17. 

Kirch  er,  3. 

Ritasato  glucose 
formate  agar, 
45. 

bouillon,  45. 
gelatin,  45. 
Klebs,  5. 

Klebs-Lroffler  ba- 
cillus, 214. 
Koch,  5. 

Kock-Week'si  ba- 
'     cillus,  221. 

Jjactases,  13. 

Latour,  4. 
Leprosy,  237. 
Lepra  bacillus, 
237. 

Leptothrix,  274. 

Leucocytic  ex- 
tract, 182. 

Ligrht  production 
by  bacteria,  17. 

Linnaeus,  3. 

Lipolytic,  13. 

Lipases,  13. 

Litmus  gelatin,  57. 
milk,  44. 
neutral  solution, 
57. 

Lower  bacteria,  24. 

Leeuwenhoek,  3. 

Lumpy  jaw,  276. 

Lustgarten's  ba- 
cillus, 237. 

Lysins,  structure 
of,  134. 


Madura  foot,  277. 

Maltases,  13. 

Malta  fever  micro- 
coccus, 201. 

Maliign*ant  oede- 
ma, 245. 

Mallein,  117. 

McConkey's  bile 
isalt  broth,  45. 

Meat   extract,  38. 

Media,  36,  37. 
agar  ag^ar,  41. 
agar  ascitic,Was- 
sermann's,  ^55. 
agar    bile  salt, 
McConkey's, 
52. 

agrar  blood, 
Washboum's, 
55. 

agar,  Braun's 
fuchsin,  53. 

agar  carbolized, 
57. 

agar  glucose,  49. 

agar  earthy- 
salts,  Lipman 
and  Brown,  49. 

ag-ar  glucose 
formate,  Kit- 
sat  o,  45. 

agrar  glycerine, 
56. 

ag'ar  Haricot,  50. 
ag-ar  Hesse-Hay- 

den-Naehrtoff, 

50. 

agar  litmus  lac- 
tose, Wurtz's, 
52. 

agar  serum,  Hei- 

man's,  56. 
agar  serum, 

'Wertheimer's 

56. 

agar  sulphindi- 
g-otate,  Weyl's, 
45. 

asparagin,  Us- 
chinsky's,  45. 

Beyrinck's  me- 
dia, 49,  50. 

bile   salt  broth, 
McConky's, 
45. 

bouillon,  41. 


bouillon  carbol- 
Lzed,  51. 

bouillon  iglucose; 

formate,  Kitas- 
ato,  45. 

bouillon  glycer- 
ine, 56. 

bouillon  glycer- 
ine, potato,  52. 

bouillon.  Hari- 
cot, 50. 

bouillon  inoslte- 
free,  Dun- 
ham's, 46. 

bouillon  litmus 
lactose,  57. 

bouillon  litmus 
lactose, 
Wurtz's,  52. 

bouillon,  Pari- 
ett's,  57. 

bouillon  serum, 
55. 

bouillon  sulphin- 

digotate, 

Weyl's,  45. 
blood  agar,  Guy's 

citrated,  42. 
blood  serum,  42. 
blood  serum, 

Councilman 
and  Mallory,  56. 
blood  serum, 

glycerine,  56. 
blood  serum,  me- 
dia, Loeffler's 

56. 

Capaldi-Pros- 
kauer    No.  1, 
47. 

Capaldi-Pros- 
kauer    No.  2, 
47. 

dextrose  bouil- 
lon, 44. 
Dorset's  egg,  43* 
egg,  43. 

egg  albumin 
agar,  55. 

egg  albumin 
broth,  Lip- 
schutz's,  55. 

filtration,  40,  60. 

fish  bouillon,  49. 

fiuid,  37. 


for  chromogenlo 
group  of  or- 
ganisms, 48. 

for  coli-typhoid 
group  of  or- 
ganisms, 51. 

for  diptheria 
bacillus,  56. 

for  diplococcus 
meningitidis, 
55. 

for  diplococcus 
pneumonia,  55« 

for  earth  organ- 
isms, 49. 

for  gonococcus, 
55. 

for  milk  organ- 
isms, 53. 

for  nitrous  or- 
ganisms, Win- 
ogradsky's,  50. 

for  nitric  organ- 
isms, Wino- 
gradsky's,  50. 

for  nitrogen  fix- 
ing organisms, 
49,  50. 

for  phosphores- 
cent organ- 
isms, 49. 

for  photogenit 
organisms,  49. 

for  plant  organ- 
isms, 50. 

for  tuberculosis 
bacillus,  56. 

for  water  organ- 
isms, 50. 

gelatin,  41. 

gelatin  carbol- 
ized,  51. 

gelatin  Eisner's 
potato,  53. 

gelatin  glucose 
formate,  Kit- 
sato,  45. 

gelatin  litmus 
lactose,  51 

gelatin  litmus 
lactose, 

Wurtz's,  52. 
gelatin  'sulphin- 

digotate, 

Weyl's,  45. 
hay  infusion,  51. 
iron  bouillon,  47. 


iron  peptone, 
Pake's,  47. 

lead  bouillon,  47. 

lead  peptone,  47. 

liquefiable,  38. 

litmus  bouillon, 
46. 

litmus  milk,  44. 
milk,  44. 
nitrate  bouillon, 
46. 

niitrate  peptone, 
47. 

peptone,  Dun- 
ham's, 44. 

potato,  43. 

potato  ;glycerin- 
ized,  57. 

proteid-free 
broth,  46. 

rice-milk,  Eisen- 
berg,  48. 

rosalic  acid  pep- 
tone, 47. 

serum  dextrose. 
Hiss,  48. 

special,  45. 

sterilization  of, 
58. 

tubing  of,  57. 
T^hey  agar,  54. 
whey  gelatin,  54. 
whey  litmus,  53. 
whey  agar,  54. 
whey  gelatin,  54. 
whey  litmus,  Pe- 
truschky's,  53. 
Measles,  281. 
Metachromic 

granules,  27. 
Metatropic  bac- 
teria, 33. 
MetchnikofC's  pha- 
gocytic the- 
ory, 177. 
Method    of  fixing 
bacteria,  72. 
of  staining  bac- 
teria, 72. 
Mesophilic  bac- 
teria, 33. 
Micrococcus  aqua- 
tilis,  310. 
candicans,  310. 
catarrhalis,  200. 
coronatus,  310. 


intracellularis 
meningitidis, 
195. 

melintensis,  201. 

pharyngis,  201. 

pneumonia,  192. 

tetragenes,  186. 

Zymogenes,  202. 
Microscopic  meth- 
od for  study 
of  bacteria, 
70,  71. 
Microspiro  coma, 
311. 

Microsporon  fur- 
fur, 280. 
Milk,  504. 

analyses,  308. 
Mobility    of  bac- 
teria, 27. 

amoeboid,  28. 

Brownian,  28. 

by  indulating 
membrane,  28. 

by  flagella. 
Mouth  flora,  8. 
Muller,  3. 
Mumps,  289. 
Mycetoma,  277. 
Mycorrhizas,  20. 

Needham,  3. 

Negri  bodies,  287. 

Neutral  volatile 
substances,  14. 

Nitration,  19. 

Nitrification,  19. 

Nitrifying  bac- 
teria, 19. 

Nitrogen  assimila- 
tion, 19. 

Nitrogen  cycle,  18. 
fixation,  20. 

Nitrosation,  19. 

Nitrosococcus,  19. 

Nocardia,  274. 

Noguchi  modifica- 
tion of  Was- 
serman,  146, 
148. 

Noma,  289. 

Nutrition  of  bac- 
teria, 15. 

Obenuier,  5. 

Obligative  aero- 
bes, 15,  85. 


anaerobes,  16,  35. 
Odium  albicans, 
280. 

Opsonins,  179. 

Opsonic  index,  180. 

Organic  acids,  14^ 

Organisms  decol- 
orized by- 
Gram's,  78. 
stained  by 

Gram's,  78. 
allied  to  cholera 
spirillum,  228. 

Origin  of  bacteria, 
5. 

Oxidizing  fer- 
ments, 13. 

FaJce's    iron  pep- 
tone, 47. 
nitrate  peptone, 
47. 

Paracolon  bacilli, 
»  265. 
group  of  organ- 
isms, 265. 
Paracliromophor- 
ousf  bacteria, 
33. 

Paratrophic  bac- 
teria, 33. 

Paratyphoid  bacil- 
lus, 266. 
group  of  organ- 
isms, 265. 

Parke  bacillus, 
269. 

Pasteur,  4. 

Pathogenic  'anaer- 
obic organ- 
isms, 240. 

Pathogenic  bacilli, 
203. 

bacteria,  32,  184. 
moulds,  278. 
properties  of 

bacteria,  105. 
yeasts,  281. 
Pathogenicity, 
method  of 
\  study,  108. 
study  of,  105. 
Pellagra,  290. 
Peptone,  Dun- 
ham's, 44. 
Pake,  47. 


PfeifCer  bacillus, 
219. 

Phagocytic  the- 
ory, 117. 

Phagolysis,  177. 

Phenol  production, 
test  for,  94.' 

Phosphorous  cy- 
cle, 22. 

Photogenic  bac- 
teria, 33. 

Pigment  produc- 
tion   of  bac- 
teria, 17. 
production,  test 
for,  95. 

Pi'tyriasies  versi- 
color, 280. 

Plasmolysis,  27. 

Plate  cultures,  60. 
study  of,  65,  68. 

Plenciz,  4. 

Plemorphism,  26. 

Pneumonia,  261. 

Pneumobacillus, 
261. 

Pneumococcus, 
192. 

Pneumoenteritis, 
255. 

Pollender,  4. 

Potatoe  media,  43. 

Precipitins,  124, 
156,  157. 

Proteid  differenci- 
ation,  150. 

Proteins  differen- 
ciation,  150. 

Protein  group  in- 
testinal bac- 
teria, 310. 

Proteolytic  fer- 
ments, 12,  32. 

Prototropic  bac- 
teria, 33. 

Pseudodiph- 

theria  bacillus, 
218. 

Pseudo  influenza, 
221. 

Pseudo  meningo- 
coccus, 201. 

Psychrophilic  bac- 
teria, 35. 

Ptomains,  118. 

Putrefaction,  12, 
32. 


Quarter  evil,  242. 

Babies,  286. 

vaccine,  168. 
Ray  fungus,  275. 
Rayer,  4. 

Reaction  of  media 
optimum,  101. 

Receptors,  124. 

Reducing  agents, 
test  for,  95. 
ferments,  14, 

Reckinghausen,  5. 

Relapsing  fever, 
297. 

Rennin,  32. 

Reproduction  of 
bacteria,  28. 

Resistance  of  leth- 
al agents,  test 
for,  101,  02. 

Respiratory  flora, 
9. 

Resting    stage  of 

bacteria,  28. 
Rinderpest,  294. 
Rindfleisch,  5. 
Ring  worm,  279. 

fungus,  279. 
Riva  test,  319. 

SaprbfTenic  bac- 
teria, 32. 

Saprophytes,  32. 

Scarlet  fever,  283. 

Scharomyces, 
Busse,  282. 

Scharomyces  tum- 
efaciens,  282. 

.Schizomycetes,  23. 

Schultz,  4. 

Schwaun,  4. 

Sensitizing  body, 
129. 

Septicaemia,  118. 
Septic  tanks  332. 
Sewage  analyses, 
332. 
bacteria,  311. 
streptococci,  311. 
Shake  cultures,  69. 
Sheath  of  bacteria, 
27. 

Shiga  bacillus,267. 
Skin  flora,  7. 
Smallpox,  285. 
vaccine,  166. 


Smear  culture,  68. 
Smegma  bacillus, 
237. 

Soft  chancre,  224. 
Soluble  engymes, 
14. 

Soor  fungus,  280, 
South  African 

horse  sickness, 

293. 

Spallanzanni,  3. 
Special  media,  45. 
Spirillaceae,  23. 
Spirillum  cholera 
asiaticae,  225. 
Deneke,  229. 
Finklei  -  Pry  or, 
229. 

Massanah,  229. 
Metchnikovi, 
228. 

Spirochetae  and 
allies,  295. 

buccalis,  296. 

calligyrum,  304. 

Carter!,  300. 

dentium,  296. 

Duttoni,  300. 

gallinarum,  298. 

macrodentium, 
304. 

mouth  of,  295. 

Obermeiri,  297. 

pallada,  300. 

pertenuis,  304. 

phagedens,  304. 

refrigens,  296. 

Vincenti,  296. 
Spore,  36. 

formation  28. 

study  of,  71. 

germination,  30. 

study  of,  72. 

resistance,  30. 
Sporulation,  28. 
Staining    of  bac- 
teria, 72. 

bacteria,  acid 
fast,  72. 

bacteria,  Mallory 
method,  86. 

bacteria,  in  tis- 
sues, 86. 

bacteria,  in  tis- 
sues, Gram- 
Wiogert  meth- 
od, 86. 


bacteria,  in,  tis- 
sues, Loefller 
method,  85. 
Staining-    of  cafp- 
sule,  81. 

capsule,  Hiss' 
method,  82. 

capsule,  John's 
method,  81. 

capsule,  McCon- 
key's  method, 
82. 

capsule,  Muir's 
method,  82. 

capsule,  Rilb- 
bert's  method, 
82. 

capsule,  Welch's 

method,  81. 
Staining  of  flagel- 

la,  83. 
flag-ella,  Bung-e's 

method^  84. 
flagella,  Loef- 

fler's  method, 

84. 

flag-ella,  Muir's 
method  modi- 
fied, Pitfeld, 
84. 

flagella,  Pit- 
feld's  (method, 
84. 

flagella,  Van  Em- 
engen's  meth- 
od, 84. 
Staining:  of  spores. 
82. 

spores,  Abbot's 
method,  83. 

spores,  double 
s^tain  method, 
82. 

spores,  Muller'is 
method,  83. 

spores,  sing-le 
stain  method, 
82. 

Staining-  techni- 
que, 77. 
technique,  dif- 
ferential, 78. 
technique,  dif- 
ferential, 
Gram's  meth- 
od, 78. 


technique,  dif- 
ferential, 
Gram's  Claud- 
ius* method, 
79. 

techniquev  dif- 
ferential. 
Gram's  negra- 
tive  method, 
78. 

technique,  dif- 
ferential, Nels- 
ser's  method, 
80. 

technique,  dif- 
ferential, 
Pappenheim's 
method,  79. 

technique,  dif- 
ferential, Zi- 
ehl  -  Nielson 
method,  79. 

technique,  ordi- 
nary, 77. 
Stains,  72. 

aniline  fuchsin 
violet,  73, 

aniline  gentian, 
73. 

alkaline  methy- 
lene blue,  73. 

alkalinei  methy- 
lene blue,  Lio- 
efller's,  73. 

alkaline  methy- 
lene blue^ 
Koch's,  73. 

Bismark  brown, 
74. 

Stains,  capsule,  81. 

capsule,  McCon- 
key's,  74. 

capsule,  Muir''s 
mordant,  75. 

capsule,  Rib- 
bert's,  75. 

carbolic  acid  so- 
lution, 73. 

contrast,  74. 

contrast.  eosin 
solutions,  74. 

contrast,  neutral 
aqueous  solu- 
tion, 74. 


contrast,  saif- 
franim  aque- 
ous solution, 
74. 

contrast,  Vesuv- 
ian  solution, 
74. 

differential,  80. 
differential. 
Gram  with 
Bismark 
brown,  80. 
differential, 
Hunt's  modi- 
fied, 80. 
differential, 

Neisser's  mod- 
ified. 80. 
differential, 
Wheal  and 
Chown,  81. 
Bhrlich's  haema- 

toxylin,  77. 
eosin  alcoholic 

solution,  74. 
eosin  aqueous 

solution,  74. 
flagella,  75. 
flageUa,  Bunge's 

mordant,  76. 
flagella,  Loef- 

fler's,  75. 
flagella,  Loef- 
fler's  mordant, 
75. 

flagella,  Muir's 

mordant,  75. 
flagella,  Pit- 

feld's,  75. 
flagella,  Pit- 

f  eld's  mordant, 

75. 

flagella.  Van  Em- 
engen's  flxing 
solutioni  76. 

flagella.  Van  Em- 
engen's  sensit- 
izing solution, 
76. 

flagella,  Van  Em- 
engen's  reduc- 
ing solution, 

Gabtoett's  acid 

blue,  74. 
Gram's  iodine 

solution,  74. 


Kuhne's  meth- 
lene  blue>  73. 

Mayer's  alum 
carmine,  77. 

Mayer's  Haema- 
tin,  77. 

Papenheim's,  76. 

Niesser's,  76. 

Nicolles  carbol- 
thionin,  76. 
Stain,  simple  ani- 
line, 73. 

special,  76. 

Spengler's,  76. 

stock  solutions, 

73. 

Unna's  borax 
methylene 
blue,  74. 

Unna's  poly- 
chrome methy- 
lene blue,  74. 

picro  -  carmine, 
77. 

Ziehl's  carbol- 
fuchsin,  74. 

Standardization  of 
media,  39. 

Staphylococcus, 
184. 

pyogenes,  184. 
pyogenes,  albus, 
186. 

pyogenes,  au- 

rens,  185. 
epidermidosis, 
albus,  186. 
Sterilization  of 

media,  58. 
Stomach   flora,  8. 
Streptococcus,  187. 
anginosus,  191. 
equinus,  191. 
fecalis,  191. 
mitis,  191. 
pneumonia,  191. 
pyogenes,  187. 
rheumaticus  of 
Poynton  and 
Paine,  192. 
Structure   of  bac- 
teria, 26. 
Sulphur  cycle,  21. 
Synthetic  activi- 
ties   of  bac- 
teria, 17. 


Temperature  opti- 
mum, test  for, 
97. 

Tetanus,  240. 

Tetanus  antitox- 
ine,  163. 

Thermal  death 
point,  35,  98, 
99,  100. 

Thermophilic  bac- 
teria, 35. 

Thiohaoteria,  24. 
Winogradsky,  16. 

Thrush,  280. 

Tinea  barbae,  279. 
cirinata,  279. 
megalosporon, 
279. 

microsporon,  279. 
syncosis,  279. 
tonsmans,  279. 

Tox  albumins,  34. 

Toxins,  34,  109, 
108,  109. 

Toxin  analysis, 
152. 

Toxoids,  153. 

Toxons,  153. 

Trachoma,  294. 

Treponema  palli- 
da, 300. 

Trichophyton,  279. 

Trypanosome,  295. 

Tube  cultures,  61. 

Tuberculins,  174. 

Tuberculosis,  229. 
avian,  235. 
bovine,  235. 

Tuberculosis  of 
cold  iblooded 
animals,  236, 

Typhoid  fever,  270. 

Typhoid  grroup  of 
organisms, 
270. 

Typhoid  vaccine, 
171. 

Typhus  fever,  284. 


Ultra  microscopic 
organisms,290. 

Uschinsky's  aspar- 
agin,  45. 
proteid  free 
t)roth,  46. 


Vaccines,  165. 

in   general,  170. 
preparation  of, 
171. 

Vegetative  stage 
of  bacteria,  28. 

Vibro  cholera,  328. 

Vibro  septique,245. 

Vincent's  angina, 
.  .296. 

Virulency  of  path- 
ogenic organ- 
isms, 105. 
of  attenuating 
or  lowering, 
106. 

of  raising  or  ex- 
alting, 105. 


Waldeyer,  5. 

Wasserman  reac- 
tion,  141,  145. 
Water,  309. 
analyses,  314. 
bacteria,  309. 
flora,  309. 
Weigert,  5. 
Weyle's  sulphindi- 
gotate  bouil- 
lon, 45. 
gelatine,  45. 
Whooping  cough, 
289. 
bacillus,  222. 
Winogradsky 's  ni- 
tric organism, 
media,  50,  377. 
nitrous  organ- 
ism, media,  50, 
377. 

silicate  jelly,338. 
Wooden  tongue, 
276. 


Yaws,  304. 
Yellow  fever,  292. 


Zoo^loea,  26. 

Zymogenic  (bacte- 
ria, 32. 


UNIVERSmr  OF  ILLINOIS-URBANA 


3  0112  069459862 


